Phosphoenolpyruvate Availability and the Biosynthesis of Shikimic

收稿日期：2013－11－20；修稿日期：2014－02－28作者简介：黄婵媛（1987－），女，硕士，研究方向为食品安全，通信地址：510110广东广州市越秀区八旗二马路38号前座301，E-mail ：hcy2011@gmail．com 。

邻苯二甲酸酯类的特性及在食品中的限量分析黄婵媛，蔡玮红，莫锡乾（广州市质量监督检测研究院，广州510110）摘要：邻苯二甲酸酯类（PAEs ）物质，作为塑料添加剂已有将近80年的历史，普遍存在于大气飘尘、工业废水、河流、土壤以及固体废弃物中，并已在食品、饮用水、人体体液中被检出，是一种全球最普遍的环境激素类污染物。

简要介绍了邻苯二甲酸酯类的特性，对国内外邻苯二甲酸酯类增塑剂在食品中的限量规定进行了分类和比较，客观分析了标准法规现状和存在的问题，并提出了建议。

关键词：邻苯二甲酸酯；特性；限量规定；标准法规中图分类号：TS201．6文献标志码：A 文章编号：1005－1295（2014）02－0066－04doi ：10．3969/j．issn．1005－1295．2014．02．017The Toxicity and Limited Provisions of Phthalate Esters in FoodHUANG Chan-yuan ，CAI Wei-hong ，MO Xi-qian（Guangzhou Quality Supervision and Testing Institute ，Guangzhou 510110，China ）Abstract ：Phthalate esters （PAEs ），as plastic additives ，have a history of nearly 80years．They are com-monly found in airborne particulates ，industrial wastewater ，rivers ，soil and solid waste ，and have been detected in food ，drinking water and body fluids．They are common worldwide environmental hormone pollutants．Ｒe-views on the characteristics of phthalates ，limited provisions and problems of domestic laws were elaborated and some suggestions were given．Key words ：Phthalate ester ；characteristic ；limited provision ；regulation0引言邻苯二甲酸酯类化合物是应用于塑料工业的主要增塑剂和软化剂，可以使塑料的柔韧性增强，容易加工，可用于工业用途［1］。

生物药剂学与药物动力学专业词汇※<A>Absolute bioavailability, F 绝对生物利用度Absorption 吸收Absorption pharmacokinetics 吸收动力学Absorption routes 吸收途径Absorption rate 吸收速率Absorption rate constant 吸收速率常数Absorptive epithelium 吸收上皮Accumulation 累积Accumulation factor 累积因子Accuracy 准确度Acetylation 乙酰化Acid glycoprotein 酸性糖蛋白Active transport 主动转运Atomic absorption spectrometry 原子吸收光谱法Additive 加和型Additive errors 加和型误差Adipose 脂肪Administration protocol 给药方案Administration route 给药途径Adverse reaction 不良反应Age differences 年龄差异Akaike’s information criterion, AIC AIC判据Albumin 白蛋白All-or-none response 全或无效应Amino acid conjugation 氨基酸结合Analog 类似物Analysis of variance, ANOVA ANOVA方差分析Anatomic Volume 解剖学体积Antagonism 拮抗作用Antiproliferation assays 抑制增殖法Apical membrane 顶端表面Apoprotein 载脂蛋白脱辅基蛋白Apparatus 仪器Apparent volume of distribution 表观分布容积Area under the curve, AUC 曲线下面积Aromatisation 芳构化Artery 动脉室Artifical biological membrane 人工生物膜Aryl 芳基Ascorbic acid 抗坏血酸维生素C Assistant in study design 辅助实验设计Average steady-state plasma drug concentration 平均稳态血浆药物浓度Azo reductase 含氮还原酶※<B>Backward elimination 逆向剔除Bacteria flora 菌丛Basal membrane 基底膜Base structural model 基础结构模型Basolateral membrane 侧底膜Bayesian estimation 贝易斯氏评估法Bayesian optimization 贝易斯优化法Bile 胆汁Billiary clearance 胆汁清除率Biliary excretion 胆汁排泄Binding 结合Binding site 结合部位Bioactivation 生物活化Bioavailability, BA 生物利用度Bioequivalence, BE 生物等效性Biological factors 生理因素Biological half life 生物半衰期Biological specimen 生物样品Biomembrane limit 膜限速型Biopharmaceutics 生物药剂学Bioequivalency criteria 生物等效性判断标准Biotransformation 生物转化Biowaiver 生物豁免Blood brain barrier, BBB BBB血脑屏障Blood clearance 血液清除率Blood flow rate-limited models 血流速度限速模型Blood flux in tissue 组织血流量Body fluid 体液Buccal absorption of drug 口腔用药的吸收Buccal mucosa 口腔粘膜颊粘膜Buccal spray formulation 口腔喷雾制剂※<C>Capacity limited 容量限制Carrier mediated transport 载体转运Catenary model 链状模型Caucasion 白种人Central compartment 中央室Characteristic 特点Chelate 螯合物Chinese Traditional medicine products 中药制剂Cholesterol esterase 胆固醇酯酶Chromatogram 色谱图Circulation 循环Classification 分类Clearance 清除率Clinical testing in first phase I期临床试验Clinical testing in second phase Ⅱ期临床试验Clinical testing in third phase Ⅲ期临床试验Clinical trial 临床试验Clinical trial simulation 临床实验计划仿真Clockwise hysteresis loop 顺时针滞后回线Collection 采集Combined administration 合并用药Combined errors 结合型误差Common liposomes, CL 普通脂质体Compartment models 隔室模型Compartments 隔室Competitive interaction 竞争性相互作用Complements 补体Complex 络合物Confidential interval 置信区间Conjugation with glucuronic acid 葡萄糖醛酸结合Controlled-release preparations 控释制剂Control stream 控制文件Conventional tablet 普通片Convergence 收敛Convolution 卷积Corresponding relationship 对应关系Corticosteroids 皮质甾体类Counter-clockwise hysteresis loop 逆时针滞后回线Countermeasure 对策Course in infusion period 滴注期间Covariance 协方差Covariates 相关因素Creatinine 肌酐Creatinine clearance 肌酐清除率Cytochrome P450, CYP450 细胞色素P450 Cytoplasm 细胞质Cytosis 胞饮作用Cytosol 胞浆胞液质※<D>Data File 数据文件Data Inspection 检视数据Deamination 脱氨基Deconvolution 反卷积Degree of fluctuation, DF DF波动度Delayed release preparations 迟释制剂Desaturation 降低饱和度Desmosome 桥粒Desulfuration 脱硫Detoxication 解毒Diagnosis 诊断Diffusion 扩散作用Dietary factors 食物因素Displacement 置换作用Disposition 处置Dissolution 溶解作用Distribution 分布Dosage adjustment 剂量调整Dosage form 剂型Dosage form design 剂型设计Dosage regimen 给药方案Dose 剂量dose-proportionality study 剂量均衡研究Dropping pills 滴丸Drug absorption via eyes 眼部用药物的吸收Drug binding 药物结合Drug concentration in plasma 血浆中药物浓度Drug Delivery System, DDS 药物给药系统Drug interaction 药物相互作用Drug-plasma protein binding ratio 药物—血浆蛋白结合率Drug-Protein Binding 药物蛋白结合Drug transport to foetus 胎内转运※<E>Efficient concentration range 有效浓度范围Efflux 外排Electrolyte 电解质Electro-spray ionization, ESI 电喷雾离子化Elimination 消除Elimination rate constant 消除速度常数Elongation 延长Emulsion 乳剂Endocytosis 入胞作用Endoplasmic reticulum 内质网Enterohepatic cycle 肠肝循环Enzyme 酶Enzyme induction 酶诱导Enzyme inhibition 酶抑制Enzyme-linked immunosorbent assays ELISA 酶联免疫法Enzymes or carrier-mediated system 酶或载体—传递系统Epithelium cell 上皮细胞Epoxide hydrolase 环化物水解酶Erosion 溶蚀Excretion 排泄Exocytosis 出胞作用Exons 外显子Experimental design 实验设计Experimental procedures 实验过程Exponential errors 指数型误差Exposure-response studies 疗效研究Extended least squares, ELS 扩展最小二乘法Extended-release preparations 缓控释制剂Extent of absorption 吸收程度External predictability 外延预见性Extraction ratio 抽取比Extract recovery rate 提取回收率Extrapolation 外推法Extravascular administration 血管外给药※<F>F test F检验Facilitated diffusion 促进扩散Factors of dosage forms 剂型因素Fasting 禁食Fibronectin 纤粘连蛋白First order rate 一级速度First Moment 一阶矩First order absorption 一级吸收First-order conditional estimation, FOCE 一级条件评估法First-order estimation, FO 一级评估法Fiest-order kinetics 一级动力学First pass effect 首过作用首过效应Fixed-effect parameters 固定效应参数Flavoprotein reductaseNADPH－细胞色素还原酶附属黄素蛋白还原酶Flow-through cell dissolution method 流室法Fluorescent detection method 荧光检测法Fraction of steady-state plasma drug concentration 达稳分数Free drug 游离药物Free drug concentration 游离药物浓度※<G>Gap junction 有隙结合Gas chromatography, GC 气相色谱法Gasrtointestinal tract, GI tract 胃肠道Gender differences 性别差异Generalized additive modeling, GAM 通用迭加模型化法Glimepiride 谷胱甘肽Global minimum 整体最小值Glomerular filtration 肾小球过滤Glomerular filtration rate, GFR 肾小球过滤率Glucuonide conjugation 葡萄糖醛酸结合Glutathione conjugation 谷胱甘肽结合Glycine conjugation 甘氨酸结合Glycocalyx 多糖—蛋白质复合体Goodness of Fit 拟合优度Graded response 梯度效应Graphic method 图解法Gut wall clearance肠壁清除率※<H>Half life 半衰期Health volunteers 健康志愿者Hemodialysis 血液透析Hepatic artery perfusion administration 肝动脉灌注给药Hepatic clearance, Clh 肝清除率Hierarchical Models 相同系列药物动力学模型High performance liquid chromatography, HPLC 高效液相色谱Higuchi equation Higuchi 方程Homologous 类似Human liver cytochrome P450 人类肝细胞色素P450 Hydrolysis 水解Hydroxylation 羟基化Hysteresis 滞后Hysteresis of plasma drug concentration 血药浓度滞后于药理效应Hysteresis of response 药理效应滞后于血药浓度※<I>Immunoradio metrec assays, IRMA 免疫放射定量法Incompatibility 配伍禁忌Independent 无关，独立Individual parameters 个体参数Individual variability 个体差异Individualization of drug dosage regimen 给药方案的个体化Inducer 诱导剂Induction 诱导Infusion 输注Inhibition 抑制Inhibitor 抑制剂Initial dose 速释部分Initial values 初始值Injection sites 注射部位Insulin 胰岛素Inter-compartmental clearance 隔室间清除率Inter-individual model 个体间模型Inter-individual random effects 个体间随机效应Inter-individual variability 个体间变异性Intermittence intravenous infusion 间歇静脉输液Internal predictability 内延预见性Inter-occasion random effects 实验间随机效应Intestinal bacterium flora 肠道菌丛Intestinal metabolism 肠道代谢Intra-individual model 个体内模型Intra-individual variability 个体内变异性Intramuscular administration 肌内给药Intramuscular injection 肌内注射Intra-peritoneal administration 腹腔给药Intravenous administration 静脉给药Intravenous infusion 静脉输液Intravenous injection 静脉注射Intrinsic clearance固有清除率内在清除率Inulin 菊粉In vitro experiments 体外试验In vitro–In vivo correlation, IVIVC 体外体内相关关系In vitro mean dissolution time, MDT vitro 体外平均溶出时间In vivo Mean dissolution time, MDT vivo 体内平均溶出时间Ion exchange 离子交换Isoform 异构体Isozyme 同工酶※<K>Kerckring 环状皱褶Kidney 肾※<L>Lag time 滞后时间Laplace transform 拉普拉斯变换Lateral intercellular fluid 侧细胞间隙液Lateral membrane 侧细胞膜Least detection amount 最小检测量Linearity 线性Linear models 线性模型Linear regression method 线性回归法Linear relationship 线性关系Lipoprotein 脂蛋白Liposomes 脂质体Liver flow 肝血流Local minimum 局部最小值Loading dose 负荷剂量Logarithmic models 对数模型Long circulation time liposomes 长循环脂质体Loo-Riegelman method Loo-Riegelman法Lowest detection concentration 最低检测浓度Lowest limit of quantitation 定量下限Lowest steady-state plasma drug concentration 最低稳态血药浓度Lung clearance 肺清除率Lymphatic circulation 淋巴循环Lymphatic system 淋巴系统※<M>Maintenance dose 维持剂量Mass balance study 质量平衡研究Masticatory mucosa 咀嚼粘膜Maximum likelihood 最大似然性Mean absolute prediction error, MAPE 平均绝对预测误差Mean absorption time, MAT 平均吸收时间Mean disintegration time, MDIT 平均崩解时间Mean dissolution time, MDT 平均溶出时间Mean residence time, MRT 平均驻留时间Mean sojourn time 平均逗留时间Mean squares 均方Mean transit time 平均转运时间Membrane-limited models 膜限速模型Membrane-mobile transport 膜动转运Membrane transport 膜转运Metabolism 代谢Metabolism enzymes 代谢酶Metabolism locations 代谢部位Metabolites 代谢物Metabolites clearance, Clm 代谢物清除率Method of residuals 残数法剩余法Methylation 甲基化Michaelis-Menten equation 米氏方程Michaelis-Menten constant 米氏常数Microbial assays 微生物检定法Microsomal P－450 mixed-function oxygenases 肝微粒体P-450混合功能氧化酶Microspheres 微球Microvilli 微绒毛Minimum drug concentration in plasma 血浆中最小药物浓度Mixed effects modeling 混合效应模型化Mixed-function oxidase, MFO 混合功能氧化酶Models 模型Modeling efficiency 模型效能Model validation 模型验证Modified release preparations 调释制剂Molecular mechanisms 分子机制Mono-exponential equation 单指数项公式Mono-oxygenase 单氧加合酶Mucous membrane injury 粘膜损伤Multi-compartment models 多室模型延迟分布模型Multi-exponential equation 多指数项公式Multifactor analysis of variance, multifactor ANOVA 多因素方差分析Multiple dosage 多剂量给药Multiple-dosage function 多剂量函数Multiple-dosage regimen 多剂量给药方案Multiple intravenous injection 多次静脉注射Myoglobin 肌血球素※<N>Naive average data, NAD 简单平均数据法Naive pool data, NPD 简单合并数据法Nanoparticles 纳米粒Nasal cavity 鼻腔Nasal mucosa 鼻粘膜National Institute of Health 美国国立卫生研究所Nephron 肾原Nephrotoxicity 肾毒性No hysteresis 无滞后Non-compartmental analysis, NCA 非隔室模型法Non-compartmental assistant Technology 非隔室辅助技术Nonionized form 非离子型Nonlinear mixed effects models, NONMEM 非线性混合效应模型Nonlinear pharmacokinetics 非线性药物动力学Non-linear relationship 非线性关系Nonparametric test 非参数检验※<O>Objective function, OF 目标函数Observed values 观测值One-compartment model 一室模型（单室模型）Onset 发生Open randomized two-way crossover design 开放随机两路交叉实验设计Open crossover randomized design 开放交叉随机设计Oral administration 口服给药Ordinary least squares, OLS 常规最小二乘法Organ 器官Organ clearance 器官清除率Original data 原始数据Osmosis 渗透压作用Outlier 偏离数据Outlier consideration 异常值的考虑Over-parameterized 过度参数化Oxidation 氧化Oxidation reactions 氧化反应※<P>Paracellular pathway 细胞旁路通道Parameters 参数Passive diffusion 被动扩散Pathways 途径Patient 病人Peak concentration 峰浓度Peak concentration of drug in plasma 血浆中药物峰浓度Poly-peptide 多肽Percent of absorption 吸收百分数Percent of fluctuation, PF 波动百分数Perfused liver 灌注肝脏Period 周期Peripheral compartments 外周室Peristalsis 蠕动Permeability of cell membrane 细胞膜的通透性P-glycoprotein, p-gp P-糖蛋白Phagocytosis 吞噬Pharmaceutical dosage form 药物剂型pharmaceutical equivalents 药剂等效性Pharmacokinetic models 药物动力学模型Pharmacokinetic physiological models 药物动力学的生理模型Pharmacological effects 药理效应Pharmacologic efficacy 药理效应Pharmacokinetics, PK 药物动力学Pharmacokinetic/pharmacodynamic link model 药物动力学-药效动力学统一模型Pharmacodynamics, PD 药效动力学Pharmacodynamic model 药效动力学模型Phase II metabolism 第II相代谢Phase I metabolism 第I相代谢pH-partition hypothesis pH分配假说Physiological function 生理功能Physiological compartment models 生理房室模型Physiological pharmacokinetic models 生理药物动力学模型Physiological pharmacokinetics 生理药物动力学模型Pigment 色素Physicochemical factors 理化因素Physicochemical property of drug 药物理化性质Physiological factors 生理因素Physiology 生理Physiological pharmacokinetic models 生理药物动力学模型Pinocytosis 吞噬Plasma drug concentration 血浆药物浓度Plasma drug concentration－time curve 血浆药物浓度-时间曲线Plasma drug-protein binding 血浆药物蛋白结合Plasma metabolite concentration 血浆代谢物浓度Plasma protein binding 血浆蛋白结合Plateau level 坪浓度Polymorphism 多态性Population average pharmacokinetic parameters 群体平均动力学参数Population model 群体模型Population parameters 群体参数Population pharmacokinetics 群体药物动力学Post-absorptive phase 吸收后相Post-distributive phase 分布后相Posterior probability 后发概率practical pharmacokinetic program 实用药代动力学计算程序Precision 精密度Preclinical 临床前的Prediction errors 预测偏差Prediction precision 预测精度Predicted values 拟合值Preliminary structural model 初始结构模型Primary active transport 原发性主动转运Principle of superposition 叠加原理Prior distribution 前置分布Prodrug 前体药物Proliferation assays 细胞增殖法Proportional 比例型Proportional errors 比例型误差Prosthehetic group 辅基Protein 蛋白质Pseudo-distribution equilibrium 伪分布平衡Pseudo steady state 伪稳态Pulmonary location 肺部Pulsatile drug delivery system 脉冲式释药系统※<Q、R>QQuality controlled samples 质控样品Quality control 质量控制Quick tissue 快分布组织RRadioimmuno assays, RIA 放射免疫法Random error model 随机误差模型Rapid intravenous injection 快速静脉注射Rate constants 速度常数Rate method 速度法Re-absorption 重吸收Receptor location 受体部位Recovery 回收率Rectal absorption 直肠吸收Rectal blood circulation 直肠部位的血液循环Rectal mucosa 直肠黏膜Reductase 还原酶Reduction 还原Reductive metabolism 还原代谢Reference individual 参比个体Reference product 参比制剂Relative bioavailability, Fr 相对生物利用度Release 释放Release medium 释放介质Release standard 释放度标准Renal 肾的Renal clearance, Clr 肾清除率Renal excretion 肾排泄Renal failure 肾衰Renal impairment 肾功能衰竭Renal tubular 肾小管Renal tubular re-absorption 肾小管重吸收Renal tubular secretion 肾小管分泌Repeatability 重现性Repeated one-point method 重复一点法Requirements 要求Research field 研究内容Reside 驻留Respiration 呼吸Respiration organ 呼吸器官Response 效应Residuals 残留误差Residual random effects 残留随机效应Reversal 恢复Rich Data 富集数据Ritschel one-point method Ritschel 一点法Rotating bottle method 转瓶法Rough surfaced endoplasmic reticulum 粗面内质网Routes of administration 给药途径※<S、T>SSafety and efficacy therapy 安全有效用药Saliva 唾液Scale up 外推Scale-Up/Post-Approval Changes, SUPAC 放大/审批后变化Second moment 二阶矩Secondary active transport 继发性主动转运Secretion 分泌Sensitivity 灵敏度Serum creatinine 血清肌酐Sigma curve 西格玛曲线Sigma-minus method 亏量法（总和减量法）Sigmoid curve S型曲线Sigmoid model Hill’s方程Simulated design 模拟设计Single-dose administration 单剂量（单次）给药Single dose response 单剂量效应Sink condition 漏槽条件Skin 皮肤Slow Tissue 慢分布组织Smooth surfaced endoplasmic reticulum 滑面内质网Soluble cell sap fraction 可溶性细胞液部分Solvent drag effect 溶媒牵引效应Stability 稳定性Steady-state volume of distribution 稳态分布容积Sparse data 稀疏数据Special dosage forms 特殊剂型Special populations 特殊人群Specialized mucosa 特性粘膜Species 种属Species differences 种属差异Specificity 特异性专属性Square sum of residual error 残差平方和Stagnant layer 不流动水层Standard curve 标准曲线Standard two stage, STS 标准两步法Statistical analysis 统计分析Statistical moments 统计矩Statistical moment theory 统计矩原理Steady state 稳态Steady state plasma drug concentration 稳态血药浓度Stealth liposomes, SL 隐形脂质体Steroid 类固醇Steroid-sulfatases 类固醇－硫酸酯酶Structure 结构Structure and function of GI epithelial cells 胃肠道上皮细胞的构造与功能Subcutaneous injections 皮下注射Subgroup 亚群体Subjects 受试者Sublingual administration 舌下给药Sublingual mucosa 舌下粘膜Subpopulation 亚群Substrate 底物Sulfate conjugation 硫酸盐结合Sulfation 硫酸结合Sum of squares 平方和Summation 相加Superposition method 叠加法Susceptible subject 易受影响的患者Sustained-release preparations 缓释制剂Sweating 出汗Synergism 协同作用Systemic clearance 全身清除率TTargeting 靶向化Taylor expansion 泰勒展开Tenous capsule 眼球囊Test product 试验制剂Therapy drug monitoring, TDM 治疗药物监测Therapeutic index 治疗指数Thermospray 热喷雾Three-compartment models 三室模型Though concentration 谷浓度Though concentration during steady state 稳态谷浓度Thromboxane 血栓素Tight junction 紧密结合Tissue 组织Tissue components 组织成分Tissue interstitial fluid 组织间隙Tolerance 耐受性Topping effect 尖峰效应Total clearance 总清除率Toxication and emergency treatment 中毒急救Transcellular pathway 经细胞转运通道Transdermal absorption 经皮肤吸收Transdermal drug delivery 经皮给药Transdermal penetration 经皮渗透Transport 转运Transport mechanism of drug 药物的转运机理Trapezoidal rule 梯形法Treatment 处理Trial Simulator 实验计划仿真器Trophoblastic epithelium 营养上皮层Two-compartment models 二室模型Two one sided tests 双单侧t检验Two period 双周期Two preparations 双制剂Two-way crossover bioequivalence studies 双周期交叉生物等效性研究Typical value 典型值※<U~Z>UUnwanted 非预期的Uniformity 均一性Unit impulse response 单位刺激反应Unit line 单位线Urinary drug concentration 尿药浓度Urinary excretion 尿排泄Urinary excretion rate 尿排泄速率VVagina 阴道Vaginal Mucosa 阴道黏膜Validation 校验Variance of mean residence time, VRT 平均驻留时间的方差Vein 静脉室Villi 绒毛Viscre 内脏Volumes of distribution 分布容积volunteers or patients studies 人体试验WWagner method Wagner法Wagner-Nelson method Wagner-Nelson法Waiver requirements 放弃（生物等效性研究）要求Washout period 洗净期Weibull distribution function Weibull分布函数Weighted Least Squares WLS加权最小二乘法Weighted residuals 加权残留误差XXenobiotic 外源物, 异生素ZZero Moment 零阶矩Zero-order absorption 零级吸收Zero-order kinetics 零级动力学Zero order rate 零级速度Zero-order release 零级释放。

Mycorrhizal manual of classical and molecular techniques1.0 Manipulation and Staining of Spores and Roots 1.1. Spore and Root Extraction from Pot Cultures/Field Samples Remove soil sample from the rhizosphere of the host plant growing in the pot with a 10-20mm diameter core borer. If the sample is taken from the field larger quantities should be sieved (100g-200g) and mixed into a 1L beaker of water before pouring through the sieves. Clay based soils will block the finer sieve quickly and care must be taken to tap the base of that sieve to encourage excess water to drain through. The same procedure used for pot culture material should then be followed:a.Wash the soil through 710µm and 45µm pore sieves with running water.b.Remove root material trapped on the 710µm sieve to check forattached mycelium of AMF with spores or for staining of roots(Trypan blue, Chlorazole Black E, Alkaline Phosphatase, Acid Fuchsinetc.) if required.c.Backwash the contents of the 45µm sieve into a small beaker. Try tokeep the volume to a minimum.a.Swirl the beaker contents and quickly decant the contents into 50mlcentrifuge tubes up to a maximum half way up the tube.b.Gently inject an equal amount of a 60% (w/v) commercial sugar(sucrose) solution into the pellet at the bottom of each tube using asyringe with a plastic tube extension. There should be a clearinterface visible between the water (above) and sugar phase (below).c.Centrifuge the capped tubes at approx. 3000 rpm for 2 minutes in abench centrifuge.d.Remove the spores caught at the interface of the two layers with thesyringe and tube attachment. Start above the interface and workdown into the sugar phase using a circular motion as some speciesproduce spores which can sink in the sugar solution while others canfloat just above the interface.e.Pour the contents of the syringe into a clean 45µm sieve, and washthoroughly to remove traces of sugar solution.f.Backwash contents into a Petri dish and view under astereomicroscope1.2. Making a permanent slide mount for reference or BEG registrationa. After extracting spores from a fresh pot culture. Isolate a minimum of 10-20 spores.b. On two clean microscope slides place one drop each of the mountant PVLG (Polyvinyllactoglycerol) and Melzer's PVLG see annex 2.Transfer half the spores to the first drop of mountant and the second half to the second drop using fine tip forceps (e.g. VOMM forceps No. 999220: HWC 118-10 Hammacher Instruments, P. O. Box 120209, D-42677 Solingen, Germany)c. Try and orientate the spores so that distinguishing features will be apparent once the coverslip is added.d. Carefully place a clean coverslip over each drop, making sure to lower the coverslip at an angle to prevent air bubbles being trapped.e. Gently apply a pressure to the coverslips of one of the slides to break open the spores. Wait 30 seconds and then apply gentle pressure in a circular motion with a soft (B) pencil to break spore walls open further (The pressure will depend on the species of AMF). This should be done under a stereomicroscope.f. If using PVLG, remember to allow the mountant to polymerise and top-up it up as necessary before sealing with clear nail varnish or white/silver car paint.g. Label the slide at one end with the species name and reference code, date, your name, and the mountant used.1.3. Histochemical Staining of Total AMF Mycelium in RootsThe presence of arbuscular mycorrhizal fungi in roots is not visible without appropriate staining. Different non-vital strains are available (eg trypan blue, chlorazole black, fuschin) to detect intraradical mycelium and they enable an estimation of the abundance of arbuscular mycorrhizal fungi within a root system (Trouvelot et al, 1986). However, they stain both dead and living fungal structures.A fuller understanding of AM functioning requires consideration of the metabolic states of both internal and external hyphae, and the relationship between these, because the physiological interactions will necessitate the presence of an active symbiotic fungus. Activity of succinate dehydrogenase (SDH), a mitochondrial enzyme, is considered as an indicator of viability of mycorrhiza but does not appear to reflect mycorrhizal efficiency for plant growth enhancement (Vierheilig & Ocampo, 1989). Alkaline phosphatase (ALP) activity, located within the phosphate-accumulating vacuoles of AM hyphae (Gianinazzi et al., 1979) has been proposed as a physiological marker for analysing the efficiency of mycorrhiza (Tisserant et al., 1993). Measurements of these two enzyme activities make it easy to directly compare the total production of fungal tissue with the proportion that is living or functional, and to compare simultaneously the production of mycelium within roots and in soil in order to determine whether (i) biomass produced in the two compartiments is interdependent and, (ii) the proportion of metabolically active hyphae differs with time.1.3.1 Root Preparationa. Wash the roots free of soil.b. Cut roots into 1cm long segments.1.3.2 Trypan blue staining of total mycelium1.Clear roots in 2% (w/v) KOH (10% can be used for verypigmented tree roots) for 15 min at 120°C in a pressure cooker(1h at 90°C in a water bath or oven) (Do not use samples thatare more than 2g.2.Rinse roots with water three times on a fine sieve or using amesh and forceps.3.Cover roots with 2% (v/v) HCl for at least 30 mins andpreferably longer.4.Throw away the HCl and cover roots with 0.05% (w/v) trypanblue in lactoglycerol (1:1:1 lactic acid, glycerol and water 5:1:1may be used if tree roots are to be stained) for 15min at 120°Cin a pressure cooker or 15min to 1h at 90°C in water bath oroven.5.Place roo t s into Petri dish with 50% (v/v) glycerol fordestaining and viewing under stereomicroscope.Figure 3with the permission of Mark Brundett1.4 Histochemical staining of active AMF mycelium in roots1.4.1 Succinate dehydrogenase (SDH)- and alkaline phosphatase (ALP) staining of intraradical hyphae1.Wash roots from soil using ice cold water and keep in ice2.Cut roots into 1cm lengths and mix the roots sample uniform3.Take two 0.2-0.5g root samples4.Clear roots in the following solution 2h at room temperature :20ml 0.05 M Tris/citric acid pH 9.250mg/ml sorbitol15 units/ml cellulase (from A. niger)15 units/ml pectinase (from A. niger).5.Rinse roots with water on a fine sieve.6.Put the roots sample into two bottles marked with SDH and ALPseparately, and add 20ml solution A and B separately7.Incubate roots pieces overnight at room temperature8.Pour out mixture solution, wash with distilled water9.Put the roots marked SDH and ALP in sodium hypochloritesolution(containing 3% and 1% active chlorine separately) 5min,then wash with distilled water10.Transfer the roots into a Peri dish11.Observe purple-black or dark-brown particles in roots undermicroscope12.Estimate root length containing stained hyphae (see section 1.5and figure 3)Solution A for SDH stainingChemical Concentration Volume(ml)Tris/HCl (pH 7.4)0.2 mol.l-15MgCl2 5 mmol.l-12NBT 4 mg.ml-15H2O6Na-succinate 2.5 mol.l-12*NBT------ Nitro-blue Tetrazonium, prepared daily.Solution B for ALP stainingChemical Concentration Volume(ml)0.05 mol.l-118mlTris/citric acid (pH9.2)1 mg.ml-120mg-naphthyl acidphosphateFast Blue RR salt 1 mg.ml-120mgMgCl20.5 mg.ml-1 1 mlMnCl2.4H2O0.8 mg.ml-1 1 ml1.5. Estimation of AMF colonisationEstimation of mycorrhizal colonization according to Trouvelot et ala. Mount 15 root fragments on one slide; prepare two slides (30 root fragments total).b. Observe these fragments under the microscope and rate according to the range of classes indicated in figure 4 and Annex 1. These classes give a rapid estimation of the level of mycorrhizal colonisation of each root fragment and the abundance of arbuscules.c. Put the values into the computer program 'Mycocalc' to calculate the parameters: %F, %M, %m, %a and %A, according to Trouvelot et al.. 1986. (see Figure 4 from Trouvelot et al 1986)o Frequency of mycorrhiza in the root systemF% = ( nb of fragments myco/total nb)*100o Intensity of the mycorrhizal colonisation in the root system M% = (95n5+70n4+30n3+5n2+n1)/(nb total)where n5 = number of fragments rated 5; n4 = number offragments 4 etc.o Intensity of the mycorrhizal colonisation in the root fragments m% = M*(nb total)/(nb myco)o Arbuscule abundance in mycorrhizal parts of root fragments a% = (100mA3+50mA2+10mA1)/100where mA3, mA2, mA1 are the % of m, rated A3, A2, A1,respectively, withmA3=((95n5A3+70n4A3+30n3A3+5n2A3+n1A3)/nbmyco)*100/m and the same for A2 and A1.o Arbuscule abundance in the root systemA% = a*(M/100)Figure 41.6. Histochemical Staining of Total and Active Soil Mycelium1.6.1. Extraction and measurement of AM fungal hyphae in soil1.Take soil cores (1x6cm) randomly from pots.2.Mix the soil sample well and then put a 2 gram sample in a 500mlbeaker.3.Suspend the soil in 250ml distilled water.4.Filter the soil suspension through a 300 m mesh sieve.5.The washings are collected and blended 30 seconds at high speed in ablender.6.Transfer the suspension to a flask, shake by hand and then stand onthe bench for one minute.7.Pipette 10ml (5mlx2) aliquots onto a millipore filter(1.0m_m pore size)and filter under vacuum using the filter holder.8.Place the filter on a microscope slide and let dry9.Stain the hyphae on the filter in lactic glycerol-trypan blue (0.05%(v/v)) for 5 minutes.10.Observe the stained filter under a coverslip at 200X magnification.11.Examine 30 random fields and estimate hyphal length by using a gridline interception method as used for evaluating mycorrhizal rootlenthes (see Figure 5 from Brundett bet al 1996).Figure5with the permission of Mark Brundett1.6.2. Estimation of succinate dehydrogenase (SDH)- and alkaline phosphatase (ALP)- active hyphae in soil1.Take soil cores and put in a beaker on ice.2.Mix the soil sample well and take two 2 gram subsamples.3.Put the sub-samples in two bottles marked with SDH and ALPseparately, and cover the soil with 20ml ice-cold water immediately4.Add 20ml incubation solution A in the bottle marked with SDH, andadd 20ml solution B in the bottle marked with ALP.5.Incubate the soil suspension at room temperature for 3 hours.6.Filter the suspension through a 300 m mesh sieve with 210mldistilled water.7.The collected washings are blended at high speed for 25 minutes.8.Transfer the suspension to a flask and leave to stand on the bench forone minute.9.Pipette 10ml (5mlx2) aliquots on a millipore filter, filter under vacuumas above.10.Transfer the filter onto a microscope slide.11.Counterstain AMF hyphae on the filter with 0.1% basic fuschin for 5minutes.12.Cover the filter with a coverslip and observe under the microscope at200X magnification.13.Estimate stained hyphal length using the gridline intersect method.Solution A for SDH stainingChemical Concentration VolumeTris/HCl(pH7.4)0.2M5mlMgCl25mM2ml*NBT4mg/ml5mlH2O 2.5M6mlNa-succinate 2.5M2ml*NBT:Nitro-blue Tetrazonium,prepared daily.Solution B for ALP stainingChemical Concentration Volume0.05M18mlTris/citric acid (pH9.2)1mg/ml20mga-naphthyl acidphosphateFast Blue RR salt1mg/ml20mg10%MgCl20.5%1ml10%MnCl20.5%1ml1.7. References∙Boddington,C.L.; Bassett,E.E.; Jakobsen,I.; Dodd,J.C. 1999 Comparison of techniques for the extraction and quantification of extra-radical mycelium of arbuscular mycorrhizal fungi in soils. Soil Biol.Biochem. 31: (3)479-482.∙Brundrett, M., N. Bougher, B. Dell, T. Grove and N. Malajczuk 1996 Working with mycorrhizas in forestry and agriculture. ACIAR Monograph 32.374. ISBN 1 86320 181 5.∙Gianinazzi S, Gianinazzi-Pearson V & Dexheimer J (1979) Enzymatic studies on the metabolism of vesicular-arbuscular mycorrhiza. 3. Ultrastructural location of acid and alkaline phosphatase activity in onionroots infected by Glomus mosseae (Nicol. & Gerd.). New Phytol 82, 127-132.∙Tisserant B, Gianinazzi-Pearson V, Gianinazzi S & Gollotte A (1993) In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections. Mycol. Res. 97,245-250.∙Trouvelot A, Kough JL & Gianinazzi-Pearson V (1986) Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthodes d’estimation ayant une signification fonctionnelle. In : Physiological andGenetical Aspects of Mycorrhizae, V. Gianinazzi-Pearson and S. Gianinazzi (eds.). INRA Press, Paris, pp.217-221.∙Vierheilig H. & Ocampo JA (1989) Relationship between SDH-activity and VA mycorrhizal infection. Agriculture, Ecosystems and Environment 29, 439-442.see these website for further informations:BEG (Banque Européenne des Glomales) websiteMark Brundrett's Working with Mycorrhizas in Forestry and Agriculture2.0 - DNA Techniques: PCR of ribosomal DNA from spores2.1. Introduction to the Polymerase Chain ReactionThe Polymerase Chain Reaction (PCR) is an in vitro technique enabling chemical amplification of DNA. With the improvement brought by the use of the heat stable Taq DNA polymerase of Thermus aquaticus and automation it is possible to obtain quick amplification even of single copy genes, starting from minute amounts of material. The impact of this technique in molecular biology is comparable to that which followed the discovery of restriction enzymes. It has been adapted for a wide variety of applications, and in particular PCR has opened the possibility to analyse organisms at the nucleic acid level even when only small amounts of nucleic acid can be obtained, as in the case of arbuscular mycorrhizal (AM) fungi. Furthermore, although the efficiency of PCR amplification is dependent on the purity of the target DNA, Taq DNA polymerase is less sensitive to template purity than other molecular biology techniques so that partially purified nucleic acid can be used. This feature is a great advantage for plant/soil microbiology research, as investigations can be made directly on partially purified biological material, like fungal spores or infected plant roots.Ribosomal genes are multicopy genes tandemly organised in the genome. Each ribosomal genes encodes for three subunits (18S[SSU], 5.8S and 28S[LSU]) separated from each other by a Inter Non Transcribed region (ITS). The genes themselves are separated from each other by an Inter Genic Spacer (IGS) (see figure).The various characteristics of rRNA and rDNA have made them a choice target for phylogenetic and taxonomic studies, and comparative studies of the nucleotide sequences in ribosomal genes has provided data for the analysis of phylogenetic relationships over a wide taxonomic range of organisms. The nucleotidic polymorphism is not evenly distributed throughout the ribosomal genes and the three regions evolve at different rates. ITS and IGS are variable regions which mutate more frequently than the three conserved coding subunit regions (18S, 5.8S, 25S). This generally makes the former more informative for analyses of closely related genomes, whereas the coding regions of the small and the large ribosomal subunit are considered to be more useful for understanding more distant relationships at the species/order level.The internal transcribed spacer region like the intergenic spacer region, evolved much faster and sequence differences between different populations of one species, or in a single spore in the case of the Glomales, can be detected. The 5' end of the large ribosomal subunit harbours two informative polymorphic domains (D1 and D2). The polymorphism observed in these domains between and in a taxa, allows also to identify specific nucleotidic sequences which can be used to design primers with different level of specificity or discrimination (van Tuinen et al 1998a).2.2. Practical aspects of PCRThe Polymerase Chain Reaction is an in vitro technique which allows the amplification of a specific region of DNA located between two known sequences. After each cycle of denaturation, annealing and extension the amount of DNA is double. Potentially, after 20 cycles of PCR, there will be a 220- fold amplification (or 1.106). This illustrates the sensitivity of thismethod, and the potential artifactual amplification of DNA, as any traces of DNA can be amplified.SCHEMATIC REPRESENTATION OF THE POLYMERASE CHAINREACTIONBefore the discovery of thermostable polymerase, DNA polymerases such as the Klenow fragment of E. coli DNA polymerase I or T4 DNA polymerase were used. Due to their heat lability, fresh aliquots of enzymes had to be added after each denaturation cycle. The first heat stable DNA polymerase (Taq polymerase) was purified from Thermus aquaticus . Today several heat stable polymerase are available, they are of natural or recombinant origin and vary in their biochemical properties such as extension rate, thermal stability, 5'?3' or 3'?5' exonuclease activity. The specificity and activity of the same enzymes is also very dependent on the producer. Some enzymes such as Tth, have a reverse transcriptase activity, they cannot therefore be used for the synthesis of cDNA.Beside the enzyme the other factors that can affect the PCR reaction are:∙Primers∙M gCl2 concentration∙Primer concentration∙Primer sequence∙Reaction stringency∙Length of the amplification product∙Number of PCR cycles∙other unknown factorsFor each PCR reaction the optimal conditions can vary depending mainly on the primer-DNA combination.The dNTP's are generally used at a concentration of 100µM, although at lower concentrations (10-100 µM) Taq polymerase has a higher fidelity. The most common buffer used with the Taq polymerase is:∙10 mM Tris/HCl pH 8,3∙50 mM KCl∙ 1.5 mM MgCl2∙0.1% (w/v) gelatinThe MgCl2 concentration affects the specificity of the PCR reaction. A too low concentration affects the final yield whereas a too high concentration reduces the specificity of the reaction. Other components often present in DNA extraction buffer can affect the enzyme activity. SDS at a concentration > 0.01% inhibits the polymerase. The inhibition of SDS (0.01%) can be reversed by some non-ionic detergents (0.5 % (v/v) Tween 20, NP 40). The primer working concentration is generally of 0.5 - 1 µM. If the primer concentration is too high primer dimerisation can occur.The primer composition is very important. In most PCR applications, the primers are designed to be exactly complementary to the template DNA. The general rules for the primer design are: a length of about 20 - 30 nucleotides. Shorter primers can be used with success and primers longer than 30 do not increase the specificity of the bindingthe GC content should be about 50%the 3' ends should not be complementary, as primer dimerisation will occur the 3' of the primer should be as homologous as possiblethe 5' can be modified to add a restriction site or a GC clamp, in this case, both primers should be equivalent in their melting temperaturesThe number of the cycles can be increased to increase the amount of product recovered, but this will also increase non-specific amplification. Beside all these factors, some primer combinations will work very well, and others not. As so many factors affect the PCR reaction it is very important to have a positive and negative control in an PCR reaction.2.3. ContaminationAs the PCR reaction is so sensitive, precautions have to be taken to avoid undesirable amplifications., such as using DNA free water and negative controls with every set of amplifications.Thermostable DNA polymerases and their sourcesDNA polymerase Natural/recombinant SourceTaq Natural Thermus aquaticusAmplitaq®Recombinant T. aquaticusAmplitaq® (StoffelRecombinant T. aquaticusfragment)Hot Tub TM Natural Thermus flavisPyrostase TM Natural T. flavisVent TM Recombinant Thermoccucus litoralisDeepVent TM Recombinant Pyrococcus GB-DTth Recombinant Thermus thermophilusPfu Natural Pyrococcus furiosusPfu Cloned Pyrococcus furiosuExo-PFU Recombinat Pyrococcus furiosuUITma TM Recombinant Thermotoga maritimaProperties of DNA polymerases commonly used in PCRTaq/ Amplitaq®StoffelfragmentVent TM Deep-VentTMPfu Tth UITma TMThermostability-half-life at 95°C40804001380>12020>505’ - 3’exonucleaseactivityYes No No No No Yes No3’ - 5’exonucleaseactivityNo No Yes Yes Yes No YesExtension rate(nt/sec)75>50>80n.i.n.i.30-40n.i.ReversetranscriptaseactivityWeak Weak n.i.n.i.n.i.Yes n.i.Resulting DNAends3’A3’A>95%blunt >95%bluntn.i.3’A BluntMolecular weight(kDa)9461n.i.n.i.929470from : PCR Newton, C.R. and Graham, A. BIOS Scientific Publishers Limited 19942.4. PCR from AMFWe present a protocol which has been used to amplify the 5' end of the large ribosomal unit of Glomales, using the fungal spore as starting material.This method can be applied to other types of biological material, like plant roots (van Tuinen et al 1998b; Jacquot et al. 2000; Turnau et al. 2001)2.4.1. Preparation of the nucleic acidsa. Collect clean and shiny Glomalean spores (1 to 10) with forceps under a binocular microscope and rinse with distilled water.b. Transfer the spores to a 1.5 ml Eppendorf tube containing 10 µl water and crush by means of a micropestle, or a glass Pasteur pipette. Disposable micropestles are available from many laboratory suppliers, and can be reused after incubation for several hours in 0.1 N NaOH to digest any remaining DNA.c. Add 30µl 100 mM Tris/HCl pH 8.0 and 10 µl of 20% Chelex 100 (Bio Rad) to the crushed spores. Vortex this suspension and then bring to 95 °C for 5 min. Cool on ice.d. Clear the suspension by centrifugation for 1 min and discard the pellet. The supernatant contains the nucleic acids for the PCR reactions. Depending on the nature of the species analysed, and especially its DNA content, the supernatant obtained can be directly used as template for PCR amplification, or be diluted up to 1/100 before use. This DNA preparation should stored at -20 °C until use.2.4.2. Preparation of the PCR reaction1.Each PCR reaction is performed in a final volume of 50 µl.2.For each set a amplification reaction a negative control, without DNA,has to be made.∙Each reaction mixture contains:For convenience and to minimise the risk of contamination, a master mixture is prepared with all the reagents except the template DNA.∙For PCR machine without heated lid, 25 - 50 µl mineral oil is laid over the mixture, and quickly spun down onto the surface.∙25 to 35 PCR cycles are performed as follows:∙Denaturation: at 95 °C for 3 minutes for the initial denaturation,93 °C and 45 seconds for the remaining cycles.∙Annealing: at 62 °C for 45 seconds.∙Extension: at 72 °C for 45 seconds (1 minute per 1 kb is an average polymerisation speed of Taq DNA polymerase).∙ A final extension of 5 minutes is performed at the end of the cycles.2.4.3. Gel electrophoresisElectrophoresis through a medium such as agarose or polyacrylamide is a standard method for the separation and purification of nucleic acids. As nucleic acids are charged molecules they will migrate when exposed to an electric field. The size of the molecules to be resolved will, influence the choice of the electrophoretic separation media. For fragments up to 500 bp, polyacrylamide gels are the most effective. Whereas for larger molecules agarose will be the medium of choice. Similar to polyacrylamide gel electrophoresis, there is a linear relationship between agarose concentration and the logarithm of the molecular weight of the DNA. Range of Separation In Gels Containing Different Amounts of AgaroseAmounts of agarose in gel in TAE (% [w/v])Efficient range of separation of linear DNA molecules (kb)0.35-600.61-200.70.8-100.90.5-71.20.4-61.50.2-32.00.1-2The migration of the DNA molecule also depends on it's conformation. The DNA molecule can be superhelical (form I), nicked circular (form II) or linear (form III). Depending on the electrophoretic conditions (ionicstrength of the buffer, intensity of the electric field) the form I can migrate faster then the linear form.Generally the DNA molecule is visualised after electrophoresis by staining with ethidium bromide (EtBr).BE CAREFUL WHEN MANIPULATING Ethidium Bromide.IT IS A POWERFUL MUTAGEN.Ethidium bromide is a fluorescent dye which intercalates between the bases of DNA. After irradiation with UV light the bound dye retransmits the light at 590 nm. Through this staining, which can be done during or after the electrophoresis, small amounts of DNA (<10 ng) can be detected.2.4.4. Nested PCR ReactionThe aim of the nested PCR reaction is to increase the specificity of the amplification reaction by performing two PCR amplifications one after the other.The first PCR reaction is performed as previously described, but for the second reaction the amplification products obtained in the first amplification cycles are used as template, after a dilution of up to 103, an internal primer.In this way the specificity of the amplification is increased as the target DNA to be amplified requires to possess the three primer binding, the efficiency of the amplification is increased as the number of cycles can be increased, without loss of specificity.Protocola. After the first PCR amplification, the reaction is checked by loading 5 µl of the amplification product on an 1.2 % agarose gel.b. For the nested PCR reaction 5µl of the amplification product diluted 500x, are used as target for the second round of amplification (25 cycles)c. The annealing temperature will depend on the primer pair used.Abbreviations:SDS: sodium dodecyl sulfatedNTP: deoxynucleosides triphosphateTAE: Tris-acetate (40 mM Tris-acetate pH 8.0 ; 1 mM EDTA)TE: Tris-EDTA (10 mM Tris/HCl pH 7.4-8.0 ; 1 mM EDTA)EDTA: ethylenediaminetetraacetate2.5. References∙Jacquot-Plumey, E van Tuinen, D Gianinazzi, S and Gianinazzi-Pearson, V 2000 Monitoring species of arbuscular mycorrhizal fungi in planta and in soil by nested PCR : application to the study of the impact of sewage sludge.Plant Soil 226, 179-188.∙Turnau, K Ryszka, P Gianinazzi-Pearson, V and van Tuinen, D 2001 Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland. Mycorrhiza 10, 169-174.∙van Tuinen, D Zhao, B and Gianinazzi-Pearson, V 1998a PCR in studies of AM Fungi: from Primers to Application.In Mycorrhiza Manual . A. K. Varma (eds) Springer-Verlag: Heidelberg , pp. 387-400∙van Tuinen, D Jacquot, E Zhao, B Golotte, A and Gianinazzi-Pearson, V 1998b Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targetednested PCR. Mol Ecol 7, 103-111.3.0 DNA Techniques: PCR-SSCP analysis3.1. IntroductionThe ultimate character that can be used to distinguish species is variation in DNA sequence between homologous genes or regions. The distinguishing。

“固定相分子筛载气归一化法微量进样器进样保留时间峰面积The stationary phase is the substance which is fixed in place for the chromatography procedure.A molecular sieve is a material containing tiny pores of a precise and uniform size that is used as an adsorbent for gases and liquids.When the mobile phase is gas, it is called eluant gas.The mobile phase is the phase which moves in a definite direction. In statistics, normalization refers to the division of multiple sets of data by a common variable in order to negate that variable's effect on the data, thus allowing underlying characteristics of the data sets to be compared.Microinjector is a kind of injector which can make injection in a very small volumn. An injector, ejector, steam ejector or steam injector is a pump-like device that uses the Venturi effect of a converging-diverging nozzle to convert the pressure energy of a motive fluid to velocity energy which creates a low pressure zone that draws in and entrains a suction fluid and then recompresses the mixed fluids by converting velocity energy back into pressure energy.Injection is a method of putting sample into the column with a syringe or injectorThe retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.The area of a peak is called peak area.色谱图 chromatogram色谱峰 chromatographic peak峰底 peak base峰高 h，peak height峰宽 W，peak width半高峰宽 Wh/2，peak width at half height峰面积 A，peak area拖尾峰 tailing area前伸峰 leading area假峰 ghost peak畸峰 distorted peak反峰 negative peak拐点 inflection point原点 origin斑点 spot区带 zone复班 multiple spot区带脱尾 zone tailing基线 base line基线漂移 baseline drift基线噪声 N，baseline noise统计矩 moment一阶原点矩γ1，first origin moment二阶中心矩μ2，second central moment三阶中心矩μ3，third central moment液相色谱法 liquid chromatography，LC液液色谱法 liquid liquid chromatography，LLC液固色谱法 liquid solid chromatography，LSC正相液相色谱法 normal phase liquidchromatography反相液相色谱法 reversed phase liquidchromatography，RPLC柱液相色谱法 liquid column chromatography高效液相色谱法 high performance liquidchromatography，HPLC尺寸排除色谱法 size exclusion chromatography，SEC凝胶过滤色谱法 gel filtration chromatography凝胶渗透色谱法 gel permeation chromatography，GPC亲和色谱法 affinity chromatography离子交换色谱法 ion exchange chromatography，IEC离子色谱法 ion chromatography离子抑制色谱法 ion suppression chromatography离子对色谱法 ion pair chromatography疏水作用色谱法 hydrophobic interactionchromatography制备液相色谱法 preparative liquid chromatography平面色谱法 planar chromatography纸色谱法 paper chromatography薄层色谱法 thin layer chromatography，TLC高效薄层色谱法 high performance thin layerchromatography，HPTLC 浸渍薄层色谱法 impregnated thin layerchromatography凝胶薄层色谱法 gel thin layer chromatography离子交换薄层色谱法 ion exchange thin layerchromatography制备薄层色谱法 preparative thin layerchromatography薄层棒色谱法 thin layer rod chromatography液相色谱仪 liquid chromatograph制备液相色谱仪 preparative liquid chromatograph凝胶渗透色谱仪 gel permeation chromatograph涂布器 spreader点样器 sample applicator色谱柱 chromatographic column棒状色谱柱 monolith column monolith column微粒柱 microparticle column填充毛细管柱 packed capillary column空心柱 open tubular column微径柱 microbore column混合柱 mixed column组合柱 coupled column预柱 precolumn保护柱 guard column预饱和柱 presaturation column浓缩柱 concentrating column抑制柱 suppression column薄层板 thin layer plate浓缩区薄层板 concentrating thin layer plate荧光薄层板 fluorescence thin layer plate反相薄层板 reversed phase thin layer plate梯度薄层板 gradient thin layer plate烧结板 sintered plate展开室 development chamber往复泵 reciprocating pump注射泵 syringe pump气动泵 pneumatic pump蠕动泵 peristaltic pump检测器 detector微分检测器 differential detector积分检测器 integral detector总体性能检测器 bulk property detector溶质性能检测器 solute property detector(示差)折光率检测器 [differential] refractive indexdetector 荧光检测器 fluorescence detector紫外可见光检测器 ultraviolet visible detector电化学检测器 electrochemical detector蒸发(激光)光散射检测器 [laser] light scatteringdetector光密度计 densitometer薄层扫描仪 thin layer scanner柱后反应器 post-column reactor体积标记器 volume marker记录器 recorder积分仪 integrator馏分收集器 fraction collector工作站 work station固定相 stationary phase固定液 stationary liquid载体 support柱填充剂 column packing化学键合相填充剂 chemically bonded phasepacking薄壳型填充剂 pellicular packing多孔型填充剂 porous packing吸附剂 adsorbent离子交换剂 ion exchanger基体 matrix载板 support plate粘合剂 binder流动相 mobile phase洗脱(淋洗)剂 eluant，eluent展开剂 developer等水容剂 isohydric solvent改性剂 modifier显色剂 color [developing] agent死时间 t0，dead time保留时间 tR，retention time调整保留时间 t&#39;R，adjusted retention time死体积 V0，dead volume保留体积 vR，retention volume调整保留体积 v&#39;R，adjusted retention volume柱外体积 Vext，extra-column volune粒间体积 V0，interstitial volume(多孔填充剂的)孔体积 VP，pore volume of porouspacking 液相总体积 Vtol，total liquid volume洗脱体积 ve，elution volume流体力学体积 vh，hydrodynamic volume相对保留值 ri.s，relative retention value分离因子α，separation factor流动相迁移距离 dm，mobile phase migrationdistance流动相前沿 mobile phase front溶质迁移距离 ds，solute migration distance比移值 Rf，Rf value高比移值 hRf，high Rf value相对比移值 Ri.s，relative Rf value保留常数值 Rm，Rm value板效能 plate efficiency折合板高 hr，reduced plate height分离度 R，resolution液相载荷量 liquid phase loading离子交换容量 ion exchange capacity负载容量 loading capacity渗透极限 permeability limit排除极限 Vh，max，exclusion limit拖尾因子 T，tailing factor柱外效应 extra-column effect管壁效应 wall effect间隔臂效应 spacer arm effect边缘效应 edge effect斑点定位法 localization of spot放射自显影法 autoradiography原位定量 in situ quantitation生物自显影法 bioautography归一法 normalization method内标法 internal standard method外标法 external standard method叠加法 addition method普适校准(曲线、函数) calibration function or curve谱带扩展(加宽) band broadening(分离作用的)校准函数或校准曲线 universalcalibration function or curve [of separation] 加宽校正 broadening correction加宽校正因子 broadening correction factor溶剂强度参数ε0，solvent strength parameter洗脱序列 eluotropic series洗脱(淋洗) elution等度洗脱 gradient elution梯度洗脱 gradient elution(再)循环洗脱 recycling elution线性溶剂强度洗脱 linear solvent strength gradient程序溶剂 programmed solvent程序压力 programmed pressure程序流速 programmed flow展开 development上行展开 ascending development下行展开 descending development双向展开 two dimensional development环形展开 circular development离心展开 centrifugal development向心展开 centripetal development径向展开 radial development多次展开 multiple development分步展开 stepwise development连续展开 continuous development梯度展开 gradient development匀浆填充 slurry packing停流进样 stop-flow injection阀进样 valve injection柱上富集 on-column enrichment流出液 eluate柱上检测 on-column detection柱寿命 column life柱流失 column bleeding显谱 visualization活化 activation反冲 back flushing脱气 degassing 沟流 channeling 过载 overloading。

C hapter 11 U se of the Yeast Two-Hybrid System to IdentifyTargets of Fungal EffectorsShunwen LuAbstractT he yeast two-hybrid (Y2H) system is a binary method widely used to determine direct interactions between paired proteins. Although having certain limitations, this method has become one of the two main systemic tools (along with affi nity purifi cation/mass spectrometry) for interactome mapping in model organisms including yeast, A rabidopsis,and humans. It has also become the method of choice for investi-gating host–pathogen interactions in fungal pathosystems involving crop plants. This chapter describes general procedures to use the GAL4-based Y2H system for identifi cation of host proteins that directly interact with proteinaceous fungal effectors, thus being their potential targets. The procedures described include cDNA library construction through in vivo recombination, library screening by yeast mating and cotransformation, as well as methods to analyze positive clones obtained from library screening. These procedures can also be adapted to confi rmation of suspected interactions between characterized host and pathogen proteins or determination of interacting domains in partner proteins.K ey words:F ungal pathogens ,P lant disease ,P rotein–protein interactions ,Y east transformation , S ynthetic dropout media ,R eporter genes ,A utoactivation ,A ureobasidin A ,a-Galactosidase assay 1.IntroductionT he yeast two-hybrid (Y2H) system was fi rst pioneered by Fieldsand Song in 1989 (1)and has been since widely used to studyprotein–protein interactions in various binary systems (2–7)and,more recently, to establish interactomes in model organisms includingyeast, A rabidopsis,and humans (8–11). This system was originallydeveloped based on the intriguing properties of the GAL4 proteinof the baker’s yeast S accharomyces cerevisiae. GAL4 is a mastertranscription activator controlling expression of several G ALgenes encoding enzymes involved in galactose utilization (12). Melvin D. Bolton and Bart P.H.J. Thomma (eds.), Plant Fungal Pathogens: Methods and Protocols,Methods in Molecular Biology,vol. 835, DOI 10.1007/978-1-61779-501-5_11, © Springer Science+Business Media, LLC 2012165166S. LuGAL4 consists of two separable and functionally essential domains:an N-terminal DNA-binding domain (BD) which binds to specifi cDNA sequences called upstream-activating sequences (UAS), anda C-terminal activation domain (AD) which interacts with basal tran-scriptional machinery (Fig. 1). In Y2H assays, the two testing proteins(the “bait” and the “prey”) are fused in frame with the BD and ADdomains, respectively, and coexpressed in a yeast strain which carriestwo or more reporter genes. A physical interaction between thebait and prey proteins would reconstitute proximity of the nativeGAL4 domains, thus activating the reporter genes (Fig. 1). Apartfrom testing the interaction between two known proteins as originallydesigned (1), Y2H can be also used for library screening to identifythe interacting partner(s) of a characterized protein (the “bait”)without prior knowledge of the nature of the “prey.” This applicationhas been adapted to identify novel targets of many functionallycharacterized proteins (8–11, 13)including proteinaceous fungaleffectors involved in plant pathogenesis (14, 15).P roteinaceous fungal effectors, e.g., avirulence (Avr) proteinsand certain host-selective toxins (HSTs), are of diverse proteins(usually small and cysteine-rich) secreted by the fungal pathogenand recognized by the host plant, in particular host–pathogeninteractions (16–18). Although specific interactions between thecharacterized fungal effectors and the corresponding host resistance(or susceptibility) genes may have been well characterized, thecognate host receptors or targets of most fungal effectors arestill unknown. It is often diffi cult to assign candidate interactingpartners for fungal effector proteins simply by exploring availableinteractomes or comparative proteomics databases because mostfungal effectors have no apparent homologies to any known proteinsMPUAS reporter geneADE2, HIS3, URA3MEL1, lacZAUR1-CF ig. 1. S chematic diagram showing the principle of the GAL4-based yeast two-hybrid system(adapted from Fields and Song (1)). A physical interaction between the bait and prey fusionproteins brings the GAL4 domains into close proximity and activates the reporter gene.U AS upstream-activating sequence; M P minimal promoter. Enzyme activities encoded bythe reporter genes: Nutritional: A DE2phosphoribosylaminoimidazole carboxylase(adenine biosynthesis); H IS3imidazoleglycerol-phosphate dehydratase (histidine biosyn-thesis); U RA3orotidine-5¢-phosphate decarboxylase (uracil biosynthesis). Colormetric:M EL1secreted a-galactosidase;lacZ intracellular b-galactosidase.Antibiotic resistance:A UR-1C mutated phosphatidylinositol: ceramide phosphoinositol transferase (conferringresistance to aureobasidin A). The specific reporter genes in related yeast strains areindicated in Table 1.16711 Use of the Yeast Two-Hybrid System to Identify Targets of Fungal Effectors( 17, 18 ) . ToxA, the major proteinaceous HST produced by the wheattan spot fungus P yrenophora tritici-repentis ( 19, 20 ) and the leaf/glume blotch fungus S tagonospora nodorum ( 21) , has been shown to interact with two chloroplast proteins, ToxABP1 (a homologue of A rabidopsis THYLAKOID FORMATION 1 protein) ( 14 ) and PCN(a plastocyanin) (15 ) . However, the host specifi city-determining target(s) of ToxA is still unknown because in both ToxA-sensitive and -insensitive wheat lines, T oxABP1 and P CN are found to be identical (100% nucleotide identity) and expressed in the samepatterns ( 14,15 ) . Furthermore, ToxA does not interact directly with T sn1 , a disease resistance-like gene which governs the ToxA-triggered susceptibility in wheat ( 22) . These fi ndings emphasize the complexity of effector–host interactions and the need for an effi cient method to unravel the interacting networks.T his chapter is intended to describe general experimental procedures to use the GAL4-based Y2H system for identifi cation of host plant proteins that directly interact with proteinaceous fun-gal effectors, thus being their potential targets. These procedures include generation of the bait construct and expression strain, cDNA library construction through in vivo recombination, library screening by yeast mating and cotransformation, as well as analysis of the library-derived positive clones. The methods described have been used successfully to identify two pathogenesis-related proteins that are differentially expressed in ToxA-sensitive and -insensitive wheat lines and interact directly with ToxA (Lu, S., Friesen, T.L., Faris, J.D., unpublished). In addition, the materials and methods used for library screening can also be adapted for confi rmation of suspected interactions between characterized host and pathogen proteins or determination of interacting domains in identifi ed protein partners. This chapter is not intended to give a comprehensive review on the rapidly evolving Y2H systems and the related protocols pub-lished during the last decades, but some key references are cited in related sections. A brief introduction on biochemical methods that can be used for further validation of physical interactions between paired proteins is given in the last section of the chapter.Plasmids and yeast strains are described using examples from the MatchMaker Y2H system (Table1 ).P lasmids used in the Y2H system act as shuttle vectors that can be maintained in E . coli and in S . cerevisiae . pGBKT7 (Fig.2a ) and pGADT7 (Fig.2b ) are the two common vectors for construction of the bait and the prey fusion proteins, respectively. pGADT7-Rec(Fig.2b ) is a prey library vector which contains a multiple cloning 2.Materials 2.1.Plasmidsand Yeast Strains2.1.1.Plasmids168S. LuT a b l e 1G A L 4-b a s e d y e a s t t w o -h y b r i d l i b r a r y s c r e e n i n g s y s t e m s aC o m p a n y Y 2H s y s t e m b f e a t u r e sG A L 4-B D p l a s m i d c s e l e c t i o n m a r k e rG A L 4-A D p l a s m i d ds e l e c t i o n m a r k e rY e a s t s t r a i n r e p o r t e r g e n e s aT h i s t a b l e i s g i v e n t o s h o w t h e a v a i l a b i l i t y o f m a j o r G A L 4-b a s e d Y 2H s y s t e m s o n l y a n d i s n o t i n t e n d e d t o r e c o m m e n d a s p e c i fi c c o m m e r c i a l s o u r c eb D e t a i l e d p r o d uc t i n f o r m a t i o n c a n b e f o u nd a t : M a t c h m a ke r , h t t p ://w w w .c l o n t e c h .c o m ; H y b r i Z A P , h t t p ://w w w .g e n o m i c s .a g i l e n t .c o m ; P r o Q u e s t , h t t p s ://w w w .i n v i t r o g e n .c o m c p G B K T 7, t h e b a i t c l o n i n g v e c t o r (F i g . 2a ); p G B K T 7-p 53 a n d p G B K T 7-L a m , t h e c o n t r o l s p l a s m i d s (C l o n t e c h ) t h a t e x p r e s s t h e t u m o r s u p p r e s s o r p r o t e i n 53 a n d l a m i n p r o t e i n , r e s p e c t i v e l y d p G A D T 7, t h e p r e y c l o n i n g v e c t o r (F i g . 2b ); p G A D T 7-R e c , t h e p r e y l i b r a r y v e c t o r (F i g . 2b ); p G A D T 7-R e c T , t h e c o n t r o l p l a s m i d (C l o n t e c h ) w h i c h e x p r e s s e s t h e S V 40 v i r u s l a r g e T -a n t i g e n k n o w n t o i n t e r a c t w i t h p 53 b u t n o t w i t h L a m16911 Use of the Yeast Two-Hybrid System to Identify Targets of Fungal Effectors site (MCS) fl anked by short stretches of sequence homologous tothe SMART (Switching Mechanism at 5 ¢ end of RNA Transcript) oligonuceotides that are designed for in vivo recombination ( 23) . Control plasmids pGBKT7-p53, pGBKT7-Lam, and pGADT7-RecTare also listed in Table1 . 1.A H109, the recipient yeast strain for expression or coexpres-sion of the fusion proteins: M ATa, trp1-901 , l eu2-3,112,ura3-52, his3-200, gal4 D , g al80 D , L YS2 :: GAL1 UAS - G AL1 TATA- H IS3, GAL2 UAS - G AL2 TATA - A DE2, URA3 :: MEL1 UAS- M EL1 TATA - l acZ, MEL1 ( s ee N ote 1 ). 2.Y 2HGold, the alternative recipient strain for expression or coexpression of the fusion proteins: M ATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4 D , gal80 D , LYS2 : : GAL1 U AS –Gal1 T ATA –HIS3, GAL2 U AS –Gal2 T ATA –ADE2, URA3 : : MEL1 U AS –Mel1 T ATA -AUR1-C, MEL1 ( s ee N ote 2 ). 3.Y 187, the library strain and a mating partner with AH109 or Y2HGold: M AT a , l eu2-3,112, ura3-52, trp1-901, his3-200,ade2-101, gal4 D , g al80 D , m et−, URA3 :: GAL1 UAS - G AL1 TATA- l acZ , M EL1 ( s ee N ote 3 ).1.10 N NaOH solution. Dissolve 40 g sodium hydroxide pellets in 80 mL of ddH 2 O . Bring volume to 100 mL with ddH 2 O . Do not autoclave. 2.10× TE buffer: 0.1 M Tris–HCl, pH 7.5, 10 mM EDTA. Adjust pH to 7.5 and autoclave. 3.10× LiAc: 1 M lithium acetate. Adjust pH to 7.5 with dilute acetic acid and autoclave.2.1.2.Yeast Strains2.2.Buffersand SolutionsHAc-Myc*I lI II/Xma I I HI IMCS I I II/Xma I HI I IIMCS F ig. 2. M ain features of pGBKT7 ( a ), pGADT7, and pGADT7-Rec ( b ) (adapted from w ).pGADT7andpGADT7-Rec are the same except that the later has the SMART III and CDS III oligonucleotide sequences ( a sterisks )which are incorporated at the MCS for in vivo recombination ( 23).P A DH1 S . cerevisiae ADH1 promoter; T A DH1 S accharomyces cerevi-siae ADH1 terminator; P T 7 T7 RNA polymerase promoter; NLS SV40 nuclear localization signal; HA HA epitope tag; c -Mycc-Myc epitope tag; A mp r Ampicillin resistance gene; K anr kanamycin resistance gene; 5 ¢ A D and 3 ¢AD pGADT7-specific primers; 5 ¢ B D and 3 ¢BD pGBKT7-specific primers.170S. Lu4.50% PEG: Resuspend 25 g polyethylene glycol (MW = 3,350) in ~20 mL ddH 2 O . Bring volume up to 50 mL with ddH 2O and sterilize through a 0.45- m m syringe fi lter ( s ee N ote 4 ).5.F ish sperm DNA (single-stranded DNA fragments): 10 mg/mL in 1× TE buffer, denatured by boiling for 10 min and immedi-ately placed on ice. Divide to aliquots in 1.5-mL microcentri-fuge tubes and store at −20°C. 6.P otassium phosphate solution (67 mM): Dissolve 0.91 g KH 2 P O 4 in ~80 mL of ddH 2O . Adjust pH to 7.5, bring volume up to 100 mL with ddH 2O , sterilize through a 0.22- m m syringe fi lter, and store at 4°C for up to 1 year. 7. L yticase stock solution: 5 units/ m L lyticase in 1× TE buffer. Store at −20°C. 8. Z -buffer: D issolve 16.1 g N a 2 H PO 4 ·7H 2 O , 5.5 g N aH 2 P O 4 ·H 2O , 0.75 g KCl, 0.246 g MgSO 4 ·7H 2 O in ~800 mL of ddH 2O . Adjust pH to 7.0, autoclave and store at room temperature for up to 1 year. 9. X -gal and X- a -gal stock solutions: Dissolve X-gal (5-bromo-4-chloro-3-indolyl- b -D -galactopyranoside) or X- a -gal (5-bromo-4-chloro-3-indolyl- a - D-galactopyranoside) in N , N dimethyl-formamide (DMF) at a fi nal concentration of 20 mg/mL. Divide to aliquots in 1.5-mL microcentrifuge tubes and store in the dark at −20°C. 10.A ureobasidin A (AbA) stock solution: 0.1 mg/mL, dissolved in 100% ethanol, stored at 4°C. 11. Z -buffer/X-gal solution: 100 mL of Z-buffer, 0.27 mL of b -mercaptoethanol, 1.67 mL of X-gal stock solution (20 mg/mL). Mix well just before use. 12. 50% glycerol (autoclaved). 13. 100% DMSO (Dimethyl sulfoxide). 14. 3 M sodium acetate (pH 4.8). 15. 95% ethanol (chilled at −20°C). 16. 0.9% NaCl solution.1.Y PD and YPDA media (for 1 L): Dissolve 10 g yeast-extract, 20 g bacto-peptone, 0.1 g adenine hemisulfate (for YPDAonly) in ~800 mL of ddH 2O . Adjust pH to 5.6–5.8 with 1 N NaOH. Add 20 g agar (for plates only) and bring volume upto 950 mL with ddH 2O . Autoclave at 121°C for 15 min. Add 50 mL of 40% glucose after cooling down to 55°C in a water bath and mix well before pouring the plates ( s ee N ote 5 ). 2.S ynthetic dropout (SD) medium (for 1 L): Dissolve 1.7 g yeast nitrogen base without amino acids and ammoniumsulfate 5.0 g ammonium sulfate, 20 gD -Glucose, 0.60–0.69 g 2.3.Yeast Media17111 Use of the Yeast Two-Hybrid System to Identify Targets of Fungal Effectorsready-to-use dropout (DO) supplement ( s ee N ote 6 ), or 100 mL of appropriate 10× DO stock solution (see below) in~800 mL ddH 2O . Adjust pH to 5.6–5.8 with 1 N NaOH. Add 20 g agar (for plates only) and bring volume to 1 L with ddH 2O . Autoclave at 121°C for 15 min. 3. 10× DO stock solution (for 1 L): Dissolve 200 mg L -Adeninehemisulfate salt, 200 mgL -Arginine HCl, 200 mg L -Histidine HCl monohydrate, 1,000 mgL -Leucine, 300 mg L -Lysine HCl, 200 mgL -Methionine, 500 mg L -Phenylalanine, 2,000 mgL -Threonine, 200 mg L -Tryptophan, 300 mg L -Tyrosine, 200 mg L -Uracil, and 1,500 mg L -Valine in 1 L ddH 2O (omit one or more amino acids as needed to make a specifi c 10× DO solution for desired SD medium, e.g., add all except forL -Leucine and L -Tryptophan to make a 10× –Leu/–Trp DO solution). Autoclave at 121°C for 15 min and store at 4°C for up to 1 year.4. S D + X- a -gal plates: Prepare appropriate SD agar plates as described above, spread 50 m L of X- a -gal stock solution onto the surface of the solidifi ed agar plates (100-mm) just before use. Alternatively, add 1 mL of X- a -gal stock solution to 1 L of SD agar medium after autoclave (cooled down to 55°C) before pouring the plates.5.S D-aureobasidin A (AbA) plates: Prepare appropriate SD agar plates as described above and add 1 mL of AbA stock solution to 1 L of SD agar medium after autoclave (cooled down to 55°C) before pouring the plates ( s ee N ote 7 ). O ptional: Kanamycin (50–100 m g /mL) can be added to all yeast media to avoid bacterial contamination.M ost strains of S . cerevisiae grow optimally at 30°C. On nonselec-tive YPD or YPDA agar plates, single cells become visible colonies usually 2–3 days after plating. On selective SD media, colonies may appear 2–5 days after plating depending on the stringency of the selection and the yeast strains ( s ee N ote 8 ). For liquid cultures starting with a single colony (2–3 mm in diameter) with shaking at 150–200 rpm, 24–36 h of incubation is suffi cient to reach stationary growth stage.F or a short-term storage, yeast cells can be maintained on agar plates (tightly wrapped with Parafi lm) and kept at 4°C for 1–2 months without losing viability ( s ee N ote 9 ). For long-term storage, yeast cells can be suspended in appropriate liquid media with 25% glycerol and kept in cryogenic vials at −80°C ( s ee N ote 10 ). To recover a3.Methods3.1.Growthand Maintenance of Yeast Strains3.1.1.General Growth Conditions3.1.2.Storageof Yeast Strains172S. Lustrain, streak a small amount of cells onto an YPD or YPDA plate and incubate at 30°C for 2–3 days. Before starting with transfor-mation or mating, verify growth phenotypes of the yeast strain by streaking the cells onto appropriate SD agar plates and allow growth at 30°C for 2–3 days.1. A mplify the gene of your interest ( G YI ) encoding the fungaleffector protein (not including the N-terminal signal peptide) by PCR using primers that contain 5 ¢ -end restriction sequencescompatible with the MCS on the bait vector pGBKT7 (Fig. 2a). Be sure that the G YI sequence to be cloned does not contain the restriction sites that have been incorporated at the 5 ¢ -ends of the PCR primers and will be translated in frame with the GAL4-BD domain. 2. P urify the amplifi ed PCR product by phenol/chloroform extraction/ethanol precipitation or column purifi cation. 3. D igest 1.0–3.0 m g of the purifi ed PCR product using the restriction enzymes incorporated into the primer sequences under the conditions described by the enzyme suppliers (inac-tivate the restriction enzymes by heating or column purifi cation after digestion).4.L igate the digested PCR product into pGBKT7 that has been linearized with compatible restriction enzymes using a T4 DNA ligase under required conditions.5. T ransform an E . coli strain with the ligated recombinant DNA(select for K anR ) and isolate the bait plasmid (pGBKT7-GYI) by column purifi cation. 6. C onfi rm the sequence identity and the correct GAL4-BD-GYI fusion by DNA sequencing using pGBKT7-specifi c primers 5 ¢ B D (5 ¢ -TCATCGGAAGAGAGTAGT-3 ¢ ) and 3 ¢ B D (5 ¢ -AGAGT-CACTTTAAAATTTGTAT-3 ¢ ) (Fig.2a ). 1.I noculate 5 mL of YPDA medium in a 50-mL centrifuge tube with a single colony (2–3 mm in diameter) of the yeast strain (AH109 or Y2HGold). Incubate overnight at 30°C with shaking (200–250 rpm). 2. I noculate 50 mL of YPDA in a 200-mL fl ask with 0.5 mL of the overnight culture from s tep 1 . Incubate at 30°C with shaking(200–250 rpm) for 3–4 h until the OD 600 = 0.4–0.6. 3.S pin down the cells in a 50-mL tube using a GSA rotor (or equivalent) at 1,000 × g for 5 min at room temperature. Discardthe supernatants and resuspend the cell pellet in sterile ddH 2 O . 4.S pin down the cells and resuspend the cell pellet in 1 mL of TE/LiAc solution (freshly prepared by mixing 5 mL of 10× TEand 5 mL of 10× LiAc with 40 mL of ddH 2O ). Use competent cells immediately to obtain best transformation effi ciency (see N ote 11 ).3.2.Generation of theBait Construct and Expression Strain3.2.1.Construction of the Bait Plasmid3.2.2.Preparationof Yeast Competent Cells11 Use of the Yeast Two-Hybrid System to Identify Targets of Fungal Effectors173B oth AH109 and Y2HGold can be used for expression of the3.2.3.Yeast Transformationbait fusion construct or coexpression of the bait and the preyconstructs. Include aureobasidin A (AbA) in SD/-LTHA and SD/-LTHA + X- a-gal plates if Y2HGold is used ( s ee N ote 7 ).1. C ombine 0.1 m g each of the bait and the prey plasmid DNA(pGADT7 or pGADT7-RecT, Table 1) in a 1.5-mL microcentri-fuge tube for each transformation experiment as listed below:(a) p GBKT7-GYI only(b) p GBKT7-GYI + pGADT7(c) p GBKT7-p53 + pGADT7-RecT (positive control)(d) p GBKT7-Lam + pGADT7-RecT (negative control)(e) N o DNA (blank control)2. A dd 0.1 mg of the denatured single-strand fi sh sperm DNA.3. A dd 0.1 mL of competent cells to each tube and mix well.4. A dd 0.6 mL of PEG/LiAc solution (freshly prepared bymixing 1 mL of 10× TE and 1 mL of 10× LiAc with 8 mL of50% PEG) to each tube and vortex at high speed for at least10 s ( s ee N ote 12 ).5. I ncubate at 30°C for 30 min (invert tube every 10 min).6. A dd 20 m L of DMSO. Mix well by gentle inversion. Do notvortex.7. I ncubate the tube in a 42°C water bath for 15–20 min.8. S pin down cells at 13,000 rpm in an Eppendorf centrifuge atroom temperature for 10 s and discard the supernatant.9. R esuspend the cell pellet in 0.5 mL of sterile 1× TE buffer.10. S pread 100 m L of the transformed cells onto SD/–Trp, SD/–Leu–Trp (-LT), and SD/–Leu–Trp–His–Ade (-LTHA) agarplates separately. Incubate the plates (face down) at 30°C for2–3 days.11. C ompare results of the fi ve transformations. The yeast trans-formants expressing the bait fusion construct alone (Expt. 1)should grow only on the SD/–Trp plate. The yeast transfor-mants coexpressing the bait construct and the pGADT7 vector(Expt. 2) should grow on the SD/-LT plate but not on theSD/-LTHA plate (Fig. 3a), like the negative control (Fig. 3c).Any growth on SD/-LTHA plate in Expt. 2 will suggest thatthe bait fusion protein has “activated” the reporter genes onits own (“autoactivation,” s ee N ote 13 ). Also, make sure thatthe colonies on the SD/-LT plate (Fig. 3a, left) in Expt. 2 aresimilar in size to those of the controls (Fig. 3b,c, left). If thecolonies are signifi cantly smaller, the expressed bait protein islikely “toxic” to the yeast, thus not suitable for further analysis( s ee N ote 14 ).174S. LuF ig. 3. S D agar plates showing the results of transformation of AH109 with a bait fusionconstruct and control plasmids. Plates were inoculated with 100 m L of the transformedcells and incubated at 30°C for 3 days. Transformants coexpressing a bait and prey plasmids( a–c, corresponding to Expts. 2–4 in Subheading 3.2.3) all grew on SD/-LT plates ( l eft,arrow indicates a single colony), but only those expressing the positive control constructs( b) were able to grow on SD/-LTHA plate ( r ight ). All transformants in Expts. 1–4 grewequally well on SD/–Trp plates which select for T RP1only (not shown). The blank controldid not produce any colonies as shown in ( d).12. I f no apparent “autoactivation” or toxicity is associated with thebait construct, pick up a single colony from the SD/–Trp platein Expt. 1 and streak onto fresh SD/–Trp plate. Incubate at 30°Cfor 2–3 days. Use the yeast cells from the streaked colonies tomake a glycerol stock and store at −80°C ( s ee N ote 10 ).13. P erform a western blot analysis to verify the expression of the fusion protein following standard procedures ( s ee N ote 15 ). T his section describes procedures for the construction of a “non-normalized” cDNA library. Key materials used for fi rst-strand cDNA synthesis and double-stranded cDNA amplifi cation are components of the “Mate & Plate” Library System (Clontech). A “normalized” cDNA library may be required if the target genes are known to be expressed at a low level. The construction of such a library involves special procedures that are not within the scope of this chapter. Existing protocols ( 24 ) may be followed if applicable. 1. G row the host plant in a greenhouse or a growth chamber under the conditions appropriate for the fungal pathosystem to be tested. 2. I noculate the plant with an effector-producing fungal isolate or infi ltrate the plant with fungal culture fi ltrates that contain the secreted effector protein, or with the purifi ed effector protein (if available). Incubate the inoculated plants for a desired period of time. 3. C ollect plant materials at several time points, e.g., 4, 12, 24, and 48 h after inoculation or infi ltration (be sure to include the point where the induced symptoms become visible). Freeze the collected materials immediately in liquid nitrogen. 4. E xtract mRNA or total RNA using standard protocols or an appropriate commercial kit ( s ee N ote 16 ). Keep the isolated RNA on ice and proceed to fi rst-strand cDNA synthesis immediately. 1. S et up the reaction in a 0.2-mL PCR tube as follows: R NA (Containing 0.025–1.0 m g mRNA or 0.1–2.0 m g totalRNA): 1–3 m LC DS III oligo(dT) primer: 1 m Ld dH 2 O : 0–2 m LT otal volume: 4 m L2. I ncubate at 72°C for 2 min, then immediately on ice for ³ 2 min.3. A dd the following to the reaction tube and then incubate at42°C for 10 min:5× First-Strand Buffer: 2.0 m L20 mM DTT: 1.0 m L10 mM dNTP Mix: 1.0 m LM MLV Reverse Transcriptase: 1.0 m L4. A dd 1.0 m L of SMART III oligonucleotide to the reactionmixture and incubate at 42°C for 1 h in a PCR thermalcycler( s ee N ote 17 ).3.3.Constructionof a cDNA Library3.3.1.Isolation of RNAfrom Plant Tissues3.3.2.First-Strand cDNASynthesis5. A dd 1.0 m L of RNase H and incubate at 37°C for 20 min. The synthesized fi rst-strand cDNA can be used immediately for PCR amplifi cation (below) or stored at −20°C for later use. 1. S et up two reactions in 0.2-mL PCR tubes, each containing as follows: F irst-strand cDNA (see Subheading 3.3.2 ): 2 m L 10× Advantage 2 PCR Buffer: 10 m L 50× Advantage 2 Polymerase Mix: 2 m L 50× dNTP Mix: 2 m L 5 ¢ PCR Primer: 2 m L 3 ¢ PCR Primer: 2 m L 10× GC-Melt Solution: 10 m L d dH 2 O : 70 m L T otal volume: 100 m L 2. R un PCR at the following conditions: 95°C, 30 s; 30 cycles of 95°C, 10 s, 68°C, 6 min; 68°C, 5 min. 3. R un 7 m L of the PCR product from each sample on a 1.0–1.5% agarose gel. A smear of range 0.3–6.0 kb should be visible (Fig. 4 , lanes 1 and 3). 1. P repare two CHROMA SPIN™ TE-400 Columns (Clontech) following the manufacturer’s instructions.2. C arefully load the double-stranded (ds) cDNA sample fromabove to the center of the gel bed’s fl at surface in each columnprepared in step 1 and centrifuge at 700 × g for 5 min.3. C ombine the fl ow-through samples (containing ds cDNA)into a 1.5-mL microcentrifuge tube.4. A dd 1/10 volume of 3 M sodium acetate (pH 4.8) and 2.5volume of 95% ethanol (prechilled at −20°C). Mix well andincubate at −20°C for 1 h to overnight.5. C entrifuge at 13,000 rpm for 20 min at room temperature.Remove the supernatant and allow the pellet in the tube to air-dry for 10 min.6. R esuspend the pellet in 25 m L of dd H 2 O .7. R un 2 m L of the purifi ed ds cDNA on a 1.0–1.5% agarose gel.The intensity of the DNA smear (Fig. 4 , lanes 2 and 4) shouldbe similar to that of the unpurifi ed ds DNA (lanes 1 and 3).The total amount of the purifi ed ds cDNA should be 2.0–5.0 m g( s ee N ote 18 ).8. P roceed to in vivo recombination with pGADT7-Rec asdescribed below or store at −20°C for later use.3.3.3.Amplification ofDouble-Stranded cDNA3.3.4.Purification ofDouble-Stranded cDNA。

8-methoxy psoralen 8-甲氧基补骨脂素electron atmosphere 电子云false positive 假阳性1,4-cineol 1，4-桉油醇10- octadecenoic acid 10-十八碳烯酸10-deacetylbaccatin 10-去乙酰基巴卡亭13-methylpalmatine 去氢延胡索素16-hydroxytriptolide 16羟基雷公藤内酯醇2,3-dibenzyl butyrolactone lignans 2,3-二苄基丁内酯木脂素2,4,6-trinitrophenol 2,4,6-三硝基苯酚2,6-dichloroquinone-4-chloroimide 2,6-二氯苯醌氯亚胺2-deoxyribose 2-脱氧核糖2-hydroxylsugar 2-羟基糖4-aminoantipyrine 4-氨基安替比林4-demethyliridoids 4-去甲基环烯醚萜4-hydroxy benzaldehyde 4-羟基苯甲醛4-hydroxymethylphenyl-b-D-glucopyranoside 4-羟甲基苯-b-D-葡萄吡喃糖苷5-HT 5-羟色胺6’-O-methylhonokiol 6’-O-甲基和厚朴酚6-epiharpagide 6-表哈帕苷7β-O-ethylmorroniside 7β-O-乙基莫诺苷9,12-octadecadienoic acid 9,12-十八二烯酸（亚油酸）9-hexadecenoic acid 9-十六碳烯酸9-octadecenoic acid 9-十八碳烯酸（油酸）Aabdominal distension 中满，腹胀abate jaundice 退黄（疸）abdominal fullness and distention 脘腹胀满abietane 松香烷abietic acid 松香酸absorbent 吸附剂acacetin 刺槐素Acacia catechu 儿茶acacias 洋槐Acanthaceae 爵床科acanthopanax 刺五加属acetal 乙缩醛acetamide 乙酰胺acetate 乙酸盐a cetate-malonate 乙酸-丙二酸acetic acid 乙酸acetic anhydride 乙酸酐acetolysis 乙酰解acetone 丙酮acetonitrile 乙腈acetyl 乙酰基acetyl acetone 乙酰丙酮acetylated 乙酰化的acetyl coenzyme A 乙酰辅酶A acetylation 乙酰化作用acetyl ginsenoside乙酰人参皂苷acetylic 乙酰化的aching of the loins and knees 腰膝酸痛Achyranthes bidentata牛膝acicular crystal 针状结晶acid 酸acid anhydride 酸酐acidic 酸的acidic hydrolysis 酸水解acidity 酸性，酸度acidulate 酸化Aconiti Radix 川乌aconitic acid 乌头酸aconitine 乌头碱acrid 辛Actions & Indications 功能主治activated charcoal 活性炭aculeatiside 颠茄皂苷acute 急性的acute lower respiratory tract infection 急性下呼吸道感染acute promyelocytic leukemia 急性髓性白血病acutif oliside 尖叶丝石皂苷acutissimin 野牡丹鞣质acutissimin A 一种复合鞣质acyclic 链状acyclic diterpenoids 无环二萜acyclic 无环的adaptogen 适应原additives 添加剂adenosine 腺苷adenylic acid 腺苷酸adhesive 黏合剂adipocyte differentiation 脂肪细胞分化adiposity 肥胖症adjacent 毗邻的adjusting immune system 调节免疫系统adjustment 调整adrenal 肾上腺的adsorbability 吸附力adsorbent 吸附剂adsorption 吸附aegle marmelos 印枸桔aerial 地上的aesculetin 七叶内酯、秦皮乙素aesculin 七叶苷、秦皮甲素ɑ-eudesmol 桉醇affinity chromatography 亲和色谱aflatoxin 黄曲霉素agar 琼脂agaropectin琼脂胶agarose琼脂糖a-glucosidase a-葡萄糖苷酶aglycone 苷元a-glycosidase a-苷酶Ailanthus altissima 臭椿ajoene 大蒜烯Akebia quinata 木通alanine dehydrase 丙氨酸脱氢酶albiflorin 白芍药苷alcohol sugar 醇糖alcoholic glycoside 醇苷alcoholic hydroxyl 醇羟基alcoholysis 醇解alcoholyze 醇解aldehyde 醛aldobionic acid 醛糖二糖酸aldohexose 己醛糖aldohexoside己醛糖苷aldose 醛糖aldoside 醛糖苷algae 藻类algae 藻类植物algin 褐藻酸alginic acid 褐藻酸alicyclic 脂环族的，脂环的Aliphatic hydrocarbons 脂肪烃aliphatic 脂肪族的aliquots 部分的Alisma orientalis泽泻alisol 泽泻萜醇alizarin 茜草素，茜草色素alkali 碱alkalify 碱化alkaline 碱的alkaloid 生物碱alkane 烷烃alkanin 紫草素alkanoids 生物碱alkyl 烷基（的），烃基（的）allergic asthma 过敏性哮喘allergies 过敏症allergy 过敏，变态反应allicin 大蒜辣素alliin 蒜氨酸alliinase 蒜氨酸酶allitridin 大蒜素Allium葱属allocryptopine 别隐品碱allomatrine 别苦参碱allose 阿洛糖allyl isosulfocyanate 异硫氰酸烯丙酯almond 杏仁almond oil 杏仁油aloe-emodin 芦荟大黄素aloin 芦荟苷alopecia areata 斑秃alpha streptococcus 甲型链球菌alphabetical 按字母表顺序的aluminum (Al) 铝aluminum trichloride 三氯化铝alzheimer dementia (AD) 老年性痴呆ambergris 龙涎香amenorrhea and dysmenorrhea 闭经痛经amethystoidin A 香茶菜甲素amide 酰胺amino acid 氨基酸amino group 氨基amino sugar 氨基糖aminobenzyl 氨基苯基aminophylline 氨茶碱ammonia氨Ammonium ceric nitrate 硝酸铈铵amorphous powders 无定形粉末amorphous solid 无定形固体amorphous 非晶型的amygdalase 苦杏仁苷酶amygdalin 苦杏仁苷amygdaloside 苦杏仁苷amyl alcohol 戊醇amylase 淀粉酶amylopectin 支链淀粉amylose 直链淀粉anabasine 八角枫碱anacardiacea 漆树科anaesthetic 麻醉anagyrine 安那吉碱，臭豆碱analgesia 镇痛analgesic 止痛剂，止痛的analgesic 止痛剂analog 类似物analogue 类似物a-naphthol a-萘酚Andrographia paniculata穿心莲andrographolide 穿心莲内酯anemia 血虚萎黄，贫血症Anemarrhena asphodeloides知母anemarrhena 知母anesthesia 麻醉anethole 茴香醚anethole 茴香脑Angelica sinensis当归angelic acid 当归酸angelicin 异补骨脂素，白芷素angiosperm 被子植物Angiosperms 被子植物angletype 角型angstroms 埃（长度单位）angular methyl 角甲基anhydride 脱水物anhydrous 无水的aniline 苯胺anion 阴离子anion-exchange 阴离子交换体anisaldehyde sulphuric acid 茴香醛-硫酸anisaldehyde 茴香醛，对甲氧基苯甲醛anisatin 莽草毒素anisic acid 茴香酸，对甲氧基苯甲酸anisodine 樟柳碱aniso-hybridization 不等性Anogeissus latifolia 宽叶榆绿木anomer 端基异构体anomeric carbon 端基碳anomeric carbon 端基异构碳ansu 山楂antagonist 拮抗剂antagonistic 拮抗的，对抗的antagonized 拮抗的anthelminthic 驱虫anthocyanidins 花青素anthocyanins 花色素anthracene 蒽anthracenol glycoside 蒽酚苷anthranol 蒽酚anthraquinone 蒽醌anthrone 蒽酮anti-aging 抗衰老antiallergic 抗过敏药antianaphylaxis 抗过敏antiangiogenic 抗血管生成anti-arrhythmia 抗心律失常antiarthritic 抗关节炎的anti-asthma 平喘作用anti-asthmatic 平喘的antibacterial 抗菌的antibacterium 抗菌antibiosis 抗菌(作用)antibiotic 抗菌素，抗生素anticancer 抗癌anticancer 抗癌的anticoagulant 抗凝剂anticoagulation 抗凝血作用anticonvulsant 抗惊厥的antidiabetic activity 抗糖尿病活性antidiabetics 抗糖尿病药antidiarrhreal 止泻的anti-fatigue 抗疲劳antifertility 抗生育的antiflammatory 抗炎的antifungal 抗真菌的antiherpesvirus 抗疱疹病毒antihyperglycemic 降高血糖的antihypertensive 降压antihypolipidemic 降血脂的anti-inflammatory 抗炎的(药) anti-inflammatory 抗炎药antimalaria 抗疟anti-microbial 抗菌的antimony chloride 氯化锑antimony trichloride 三氯化锑anti-mutagenic 抗突变anti-nociceptive 镇痛antioxidant 抗氧化antioxidant 抗氧化，抗氧剂antioxidative 抗氧化的antipyretic 退热剂anti-radiation 抗辐射antirheumatic 抗风湿剂antisepsis 抗菌，防腐，消毒antiseptic 消毒antiseptics 防腐剂anti-stress应激antithrombin 抗凝血酶antitumor 抗肿瘤antitussive 止咳药antiulcer 抗溃疡的antivenin 抗蛇毒素antiviral 抗病毒的Antivirus 抗病毒apatites 磷灰石apiaceae 伞形科apigenin 芹菜素apiose 芹菜糖Apocynaceae 夹竹桃科apoptosis 细胞凋亡apparatus 容器approximation 近似值apricot 杏，杏仁，杏树aqueous 水的aqueous solution 水溶液arabinogalactan 阿拉伯半乳糖arabinogalactan 阿拉伯半乳聚糖arabinose 阿拉伯糖arabinosyl阿拉伯糖基Araceae 天南星科arachic acid 花生酸，二十烷酸arachidic acid 花生酸arachidonic acid 花生四烯酸Arachis hypogaea 落花生Araliaceae 五加科Aralia chinensis楤木arbutin 熊果苷arctigenin 牛蒡子苷元Arctii Fructus 牛蒡子arctiin 牛蒡子苷Arctium lappa 牛蒡arctostaphylos uva-ursi 熊果areca seeds 槟榔arecatannin 槟榔鞣质arecolidine 槟榔碱arginine 精氨酸armeniacae semen amarum 苦杏仁Armillaria mellea 蜜环菌arnidiol 阿里二醇aroma 香气，芳香气aromatic ring 芳香环aromaticity 芳香性arrow-root木薯arsenic 砷arsenic trioxide 三氧化二砷arteannuic acid 青蒿酸arteether 蒿乙醚artelinic acid 青蒿中的倍半萜artemether 蒿甲醚artemisane 艾蒿烷型Artemisia annua黄花蒿（青蒿）Artemisia capillaries thunb 茵陈蒿artemisia ketone 蒿酮Artemisia scoparia waldst. et kit. 茵陈artemisiae scopariae herba （yinchen）茵陈artemisinin 青蒿素artesunate 青蒿琥酯artesunic acid 青蒿琥珀酸arthralgia 关节痛articulation 关节artifact 人工产物arundoin 芦竹素asarone 细辛醚ascaridole 驱蛔素Asclepiadaceae 萝藦科ascorbic acid 抗坏血酸，维生素Cash灰分asparagines 天冬酰胺aspartic acid 天冬氨酸asperuloside 车前草苷asphaltene 沥青质assay 试验Asteraceae 菊科Asteropus sarasinosum 海棉Aster tataricus 紫菀asthma 哮喘Astragali Radix黄芪astragalin 紫云英苷astragaloside黄芪苷 / 黄芪甲苷astragalus 黄芪属Astragalus gummifer 胶黄芪Astragalus membranaceus 膜荚黄芪Astragalus membranaceus(Fisch.) Bge.var.mongholicus蒙古黄芪astringency 收敛astragenol 黄芪醇astringent 收敛剂asymmetric 不对称的atherosclerosis 动脉粥样硬化atom 原子atractylone 苍术酮atropa belladonna 颠茄atropine 阿托品atropine 阿托品Aurones 橙酮authentic crude drug 标准药材auxochrome 助色团auxtochrous group 助色团availability 可利用性axial bond 直立键azelaic acid 壬二酸azulenoids 奥类化合物Bbaccatin 巴卡亭Bacillus aeruginosus 绿脓杆菌Bacillus coli 大肠杆菌Bacillus typhi 伤寒杆菌bacterial dysentery 细菌性痢疾bacteriological 细菌学的bacteriostat 抑菌剂bael 印度枳baicalein 黄芩素baicalin 黄芩苷bakuchalcone 补骨脂呋喃查耳酮bakuchiol 补骨脂酚baptifoline 野靛叶碱，膺靛叶碱barbaloin 芦荟苷barbary wolfberry fruit 枸杞子barium钡barium hydroxide 氢氧化钡basic solution 碱溶液bassorin 黄芪胶糖batches 成批,分批bavachalcone 补骨脂查耳酮bavachinin 补骨脂甲素甲醚bavachromanol 补骨脂色酚酮bavachromene 补骨脂色烯查尔酮be bound up 与…… 连在一起be indigenous to 土生土长bearberry leaves 熊果叶belladonna 颠茄Benefiting补益benzaldehyde 苯甲醛benzene 苯benzene hexachloride 六六六，六氯化苯，六氯环己烷benzoic 苯甲酸的benzoquinone 苯醌benzylpaeoniflorin 苯甲酰芍药苷berbamine 小檗胺berberastine 5-羟基小檗碱berberidaceae 小檗科berberine 小檗碱berberrubine 小檗红碱bererine 黄连素betain 甜菜碱betaine 甜菜碱Betulaceae 桦木科Betulin 白桦酯醇betulinaldehyde 白桦酯醛Betulinic acid 白桦酯酸betulinicacid 白蜡脂酸bicuculline 荷包牡丹碱bicyclic diterpenoids 双环二萜bicyclic sesquiterpenoids 双环倍半萜bicyclic 双环的bidirectional双向的Biflavonoids 双黄酮类Bignoniaceae 紫葳科bile acid 胆汁酸biliary 胆汁的bilobalide 白果内酯bilobalide 银杏内酯Bilobetin 白果素binding agent 粘合剂bioactive action 生物活性作用biogenetic 生源的biological source 基原biomass 生物体biosynthesis 生物合成biosynthetic 生物合成的biotransformation 生物转化bisabolane 没药烷bisdesmosides 双糖链苷bisdesmosidic saponins 双糖链皂苷bitter 苦味bitter apricot seed 苦杏仁bitter gourd 苦瓜bitter principle 苦味素bitter principles 苦味素bitterness 苦味black soyabean spermoderm 黑大豆皮bleaching powder 漂白粉blistering 发泡blood deficiency and sallow yellow 血虚萎黄blood ejection and spontaneous external bleeding 吐血衄血blood stasis and amenorrhea 瘀血经闭Blumea balsmifera艾纳香body fluid 体液boiling water 沸水boraginaceae 紫草科borax type 硼酸型boric acid 硼酸borneol 冰片，龙脑borneol 龙脑bornylmagnolol 龙胞基厚朴酚boron trifluoride 三氟化硼bottleneck瓶颈bracken fern 羊齿植物bradycardic 减慢心率的branched-chain 支链brassia alba白芥子brassia nigra黑芥子breast 乳腺bridging atom桥接原子bromelin 菠萝蛋白酶bromine 溴bromo 溴代bromophenol blue 溴酚蓝bronchial asthma 支气管哮喘bronchial smooth muscle 支气管平滑肌bronchitis 支气管炎bronchus 支气管bronic acid 硼酸brucine 马钱子碱bufadienolide 蟾蜍甾二烯内酯buffer medium 缓冲介质building block 组成…...的基本单位Bulbus Allii 大蒜Bupleuri Radix 柴胡Bupleurum chinense柴胡Bupleurum scorzonerifolium 狭叶柴胡Burseraceae 橄榄科butanol 丁醇butanone 丁酮butyl acetate 乙酸丁酯butyl alcohol 丁醇butylidene 丁烯基C cadinene 荜澄茄烯cadinane 杜松烷cadinol 杜松醇cadmium 镉caffeetannins 咖啡鞣质caffeic acid 咖啡酸caffeotannic acid 绿原酸caffeoyl 咖啡酰基calcium carbonate 碳酸钙calcium hypochlorite 次氯酸钙calibration 校准calm panting and suppress cough 止咳平喘caloglossa 鹧鸪菜calycosin 毛蕊异黄酮camelliatannin 山茶素，山茶鞣质Campanulaceae 桔梗科campesterol 菜油甾醇camphane 莰烷型camphene 莰烯camphor 樟脑camphoric acid 樟脑酸camptothecine 喜树碱candidate 候选者cannabinoid 大麻素canophyllal 海棠果醛canophyllic acid 海棠果酸cantharidin 斑蝥素cantoniensistriol 广东相思子三醇carabranecarane 蒈烷型carbocation 碳正离子carbohydrate 碳水化合物carbon 碳carbon tetrachloride 四氯化碳carbonium ion 正碳离子carbonyl 羰基carbonyl group 羰基carboxyl 羧基carboxyl methyl 羧甲基carboxylate 羧酸盐(酯)carboxylic acid 羧基？？羧酸？？？？carbuncle 痈carcinogenic 致癌的cardenolide 强心甾烯内酯cardiac contractility 心肌收缩性cardiac glycoside, cardioactive glycosides 强心苷cardiac output 心输出量cardio and cerebral vascular 心脑血管cardiodynia 心痛，胸痛cardio-protective 保护心脏Cardioprotector 心脏保护剂cardiovascular 心血管的cardiovascular and cerebrovascular diseases 心脑血管疾病carminative 祛风剂carica papaya 番木瓜carotene 胡萝卜素carotenoid 类胡萝卜素carrageenan 角叉菜胶Carthamin 红花苷Carthamone 醌式红花苷Carthamus tinctorius 红花carvone camphane 葛缕樟烷carvone camphor 香芹樟脑carvone 藏茴香酮Caryophyllaceae 石竹科caryophyllene 丁香油烃caryophyllene 石竹烯cassia obtusifolia 决明子cassia oil 肉桂油cassiamine 山扁豆双醌castalagin 栗木鞣花素casticin 紫花牡荆素casuarictin 木麻黄鞣亭casuarinin 木麻黄鞣宁catalpol 梓醇catalysis 催化作用catalyze 催化cataract 白内障Catechin 儿茶素catechol 儿茶酚catechu 儿茶catechuic acid 儿茶酸，儿茶素category 类别cation阳离子cation-exchange 阳离子交换体Celastraceae 卫矛科cellulase 纤维素酶cellulose 纤维素central excitation 中枢兴奋central nervous system中枢神经系统centrifuge 离心centrifuging-sedimentation 沉降法cephalin 脑磷脂cephalotaxin 三尖杉碱ceratonia siliqua 长角豆cerebral ischemia 脑缺血cervical cancer 子宫颈癌C-glycosides 碳苷C-glycosidic ellagitannins C-苷逆没食子鞣质chalcone 查耳酮chamigrene 花柏烯charcoal 炭chebuloyl (Che) 诃子酰基chemical ionization 化学电离chemical ionization (CI) 化学电离chemical shift 化学位移chemisorption 化学吸收作用chenodeoxycholic acid, chenodiol 鹅去氧胆酸chenopodium 土荆芥油chestnut 栗子Chinese galls 五倍子Chinese herbal medicine 中草药Chinese magnoliavine 五味子Chinese patent medicine 中成药Chinese sumac 盐肤木chiral carbon 手性碳chirality 手性chitin 几丁质cholesterol 胆固醇chloride 氯化物chloroform 氯仿, 三氯甲烷chlorogenic acid 绿原酸chlorophyll 叶绿素chlorophyll 叶绿素chloroquine 氯喹cholagogue 利胆的choleresis 利胆choleretic 利胆剂cholesterol 胆固醇, 胆甾醇chondroitin sulfate 硫酸软骨素chondrus cryspus 皱波角叉菜chorioepithelioma 绒癌chromatography 色谱chromic acid 铬酸chromogenic 显色的，产色的Chromone 色原酮chromophore 发色团，生色团chromophoric group 发色基团chronic infantile convulsions 小儿慢惊风chronic liver disease 慢性肝病chronic nephritis 慢性肾炎chronic toxicity 慢性毒性chronic tracheitis 慢性气管炎chrysophanol 大黄酚chuanliansu,toosendanin 川楝素cinchona 金鸡纳树皮cineole 桉叶油素cineole, eucalyptol 桉油精cinnabar 朱砂cinnabaris 辰砂cinnamaldehyde 桂皮醛cinnamic acid 桂皮酸Cinnamomum cassia肉桂cinnamon bark 肉桂皮cinnamon 肉桂cinnamyl acetate 乙酸肉桂酯cinnantannincinncassiol 肉桂萜醇cinnzeylanine 乙酰桂二萜醇cinnzeylanol 桂二萜醇circular dichroism 圆二色性cirrhosis 肝硬化cis顺式的Cistaceae 半日花科citral 柠檬醛Citri Reticulatae Pericarpium 陈皮citric acid 柠檬酸citronella oil 香茅油citronellol 香茅醇citrous 柑橘属植物的citrus aurantium枳壳Citrus aurantiun 酸橙citrus limonis 柠檬树Citrus peels 橙皮Citrus reticulata橘citrus 柑橘类植物classification 分类clear and benefit the head, eyes, and throat 清利头目，利咽clear fever from deficiency 清虚热clear heat and cool the blood 清热凉血clear heat and resolve fire toxicity 清热解毒clear-cut 清晰的cleavage 裂解clematis 铁线莲clerodane 克罗烷clinic 临床上cloud density 电子云密度clove 丁香coagulation 凝血coarse polysaccharides ch in eesis (CPSC) 五味子多糖coarse powder 粗粉coating 包衣，涂料cocaine 可卡因cocarcinogenic 辅致癌的codeine 可待因coefficient 系数Coelenterate 腔肠动物coenzyme 辅酶cognitive 认知的colchicine 秋水仙碱cold fever 感冒发热collision 碰撞colloidal state 胶体状态colon bacillus 大肠杆菌colorectal cancer 结肠直肠癌colubrine 克鲁勃林columbamine 非洲防己碱column chromatography 柱色谱法column 柱coma 昏迷combretaceae 使君子科common camptotheca root 喜树根common coltsfoot flower 款冬花complex crystal 络合物结晶complex enzyme 复合酶complex tannins 复合鞣质compositae 菊科comprehensive 综合的compromise 折中方法，综合各因素方法concentrate 浓缩concentration 浓度concentrated acid 浓酸condensation reaction 缩合反应condensed tannins 缩合鞣质condenser 冷凝器confer on 授予configuration 构型conformation 构象conical 园锥形的conifer 松柏类Coniine 毒芹碱conjugate acid 共轭酸conjugate 共轭conjugated effect 共轭效应conjugated system 共轭系统consecutive reaction 连续反应constipation 便秘constipation due to deficiency of blood and body-fluid 肠燥便秘constrain the lung and stabilize panting 敛肺平喘contraceptive 避孕的，避孕药contractility 收缩性，收缩力conventional 传统的，习惯的convergence 收敛，会聚cool the blood 凉血cooling 清凉co-polymerization 异分子聚合copper 铜Coptidis Rhizoma 黄连coptisine 黄连碱coriamyrtin 马桑毒素corilagin 柯里拉京，鞣云实精Cornus officinalis 山茱萸coronary 冠状动脉coronary artery 冠状动脉coronary heart disease 冠状动脉心脏病correctant 矫味剂correlation spectroscopy 化学位移相关光谱cortex 皮质cortical皮层的，皮质的corybulbine 紫堇球碱，紫堇鳞茎碱corydaline 紫堇碱Corydalis Rhizoma 延胡索corydalmine 紫堇单酚碱corylidin 双羟异补骨脂定corynebacterium diphtheriae 白喉杆菌cosmetics 化妆品cough and asthma 咳嗽气喘cough and copious phlegm 咳嗽痰多cough-supressing and dispelling phlegm 止咳祛痰coumarin 香豆素coumarin glycoside 香豆素苷coupling constant 耦合常数course wind and dissipate heat 疏散风热covalent bond 共价键coexist 共存crab 蟹cranial nerve 脑神经crataegus pinnatifida山楂crimson tongue and polydipsia 舌绛烦渴criteria 标准critical point 临界点cross-conjugated 交叉共轭的cross-linking 交联croton 巴豆crotonic acid 巴豆油酸croton oil 巴豆油croton tiglium巴豆crotonoside 巴豆苷Cruciferae 十字花科cryptoxanthin 隐黄质crystal 结晶体crystallization 结晶化Cucurbitaceae 葫芦科cucurbitacin Ⅰa 雪胆甲素cucurbitacin Ⅱb 雪胆乙素cucurbitacins 葫芦素类cucurbitane 葫芦素烷cucurbitine 南瓜子氨酸cultivated species 栽培种Cupressaceae柏科Cupresuflavone 柏黄酮Curcuma kwangsiensis广西莪术Curcuma phaeocaulis蓬莪术Curcuma wenyujin温郁金curcumenolactone 莪术酮内酯curcumenone 莪术酮curcumin 姜黄素curcumol 莪术醇curdione 莪二酮cyamopsis tetragonolobus 瓜尔豆cyanin 花色素苷cyanogenic glycoside 氰苷cyclanoline 轮环藤酚碱cyclase 环化酶cyclic 环状的Cyclization 环合cyclization 环化作用cycloalliin 环蒜氨酸cycloartane 环木菠萝烷 / 环阿屯烷cycloastragenol 环黄芪醇cycloeudesmol 环桉醇cyclogeraniane 环香叶烷型cycloheptatriene cation 环庚三烯正离子cyclohexane 环己烷cyclohexane 环己烷cyclohexene环己烯cyclohexylamines 环己胺cyclopentane 环戊烷cyclopentanoperhydrophenanthrene 环戊烷骈多氢菲cyclopropane 环丙烷cylindrical 园柱形的cylindrin 白茅素cymarose 加拿大麻糖Cyperaceae 莎草科cysteine sulfoxides 半胱氨酸亚砜cystine 胱氨酸cytidine 胞苷cytisus 金雀花cytotoxicity 细胞毒性Ddaidzein 大豆苷元daidzin 大豆苷，黄豆苷（异黄酮苷）dammarane 达玛烷damp-heat jaundice 湿热黄疸dampnessdaphane 瑞香烷Daphne genkwa芫花daphnetoxin 瑞香毒素daphnin 瑞香苷dark plum fruit 乌梅Daturae Flos 洋金花daturameteloside 洋金花苷dauco sterine 胡萝卜苷daucosterol 胡萝卜甾醇，胡萝卜苷daurian rhododendron oil 满山红油dauricine 蝙蝠葛碱decanoylacetaldehyde 癸酰乙醛decanta 泻下decarboxylate 脱羧decoction 煎煮decompose 分解decomposition 分解，变质decompression 降压decyl 癸基defatted 脱脂degradation 降解dehydration 脱水dehydrocholic acid 脱氢胆酸dehydrocorybulbine 去氢紫堇鳞茎碱dehydrocorydaline 去氢紫堇碱dehydrogenate 脱水dehydrogenation 脱氢作用dehydroglaucine 去氢海罂粟碱dehydrohexahydroxydiphenoyl (DHHDP) 脱氢六羟基联苯二甲酰基dehydronantenine 去氢南天宁碱20(21)-dehydroxylation-ginsenoside 20(21)-脱羟基-人参皂苷deionise 去离子dementia 痴呆demethylation ephedrine 去甲基麻黄碱demulcent 镇痛剂dencichine 三七素dendrobine 石槲碱dense 粘稠的，稠密的deodorant 除臭剂deohydroandrographolide 脱水穿心莲内酯deoxy 脱氧的deoxyandrographolide 去甲穿心莲内酯deoxycholic acid 去氧胆酸deoxyschisandrin 五味子甲素deoxysugar 去氧糖deprotonation 去质子化作用depside 缩酚酸derivative 衍生物，派生物dermatitis 皮炎dermatosis 皮肤病dermerethistica 皮肤刺激药Derris elliptica 毛鱼藤deshielding去屏蔽desinsection 杀虫determination 决定，鉴定detoxify 解毒detrimental 有害的，不利的detumescence 消肿deuterium 氘，重氢development system 展开系统dexiotropic 右旋的dextrorotation 右旋D-galacto-D-manoglycon D-半乳糖-D-甘露糖基D-galactouronic acid D-半乳糖醛酸diabetes 糖尿病diabetes caused by internal heat 内热消渴diacolation 渗滤dialcol 二醇dialdehyde 二醛dialysis 透析diameter 直径diarrhea 腹泻diastereoisomer 非对映异构体diastolize 舒张diazomethane 重氮甲烷diazonium 重氮化合物dibenzyl-etrahydroisoquinolin 双苯甲基四氢异喹啉类dichlorodiphenyltrichloroethane二氯二苯三氯乙烷，滴滴涕dichloromethane 二氯甲烷dicotyledon 双子叶植物diels-alder (D-A反应）双烯加成反应diene二烯（烃）dietary fiber 膳食纤维diethyl ether 乙醚diethylaminoethyl 二乙氨基乙基diffraction 衍射digestive system 消化道系统diginatigenin 双羟基洋地黄毒苷元digitalose 洋地黄糖digitoxigenin 洋地黄毒苷元digitoxin 洋地黄毒苷digitoxose 洋地黄毒糖digoxigenin 异羟基洋地黄毒苷元digoxin 地高辛dihydrochalcones 二氢查耳酮类dihydroflavonol 二氢黄酮醇dihydromorin 二氢桑色素dihydroquercetin 二氢槲皮素dihydrosanguinarine 二氢血根碱dilactone 双内酯dilatation 扩张dilate 扩大，膨胀dilute acid 稀酸dilute alcohol 稀醇dilute alkali solution 稀碱液dilute base 稀碱Dimeric 二聚的dimers 二聚体dimethoxy 二甲氧基dimethyl formamide 二甲基甲酰胺dimethyl sulphate 硫酸二甲酯dimethyl sulphoxide 二甲基亚砜dimethylallyl pyrophosphate 二甲基烯丙酯p-dimethylamino benzaldehyde 对二甲氨基苯甲醛dimethylaniline 二甲基苯胺dimethylformamide 二甲基甲酰胺dimethylpolysiloxane 二甲硅油dimethyltetrandrine 二甲基粉防己碱Dioscoreaceae 薯蓣科Dioscorea 薯蓣属Dioscorea nipponica 穿龙薯蓣Dioscoreae Nipponicae Rhizoma 穿山龙dioscin 薯蓣皂苷diosgenin 薯蓣皂苷元diosmin 香叶木苷Diospyros柿属Diospyros kaki柿子dipiperitylmagnolol 双辣薄荷基厚朴酚diploptene 里白烯dipole-dipole interactions 偶极偶极相互作用disaccharide 二糖discrete 分离的，不相关联的disintegrate 裂解disintegrating agent 崩解剂d-isomer 右旋异构体dispel wind and relieve pain 祛风止痛disperse accumulations and relieve pain 消积止痛disperse swelling 消肿disperse 分散，散开dissolved 溶解dissolution 溶解distending pain胀痛distention and oppression in the chest and rib-side 胸胁胀闷Distortion enhancement by polarization transfer (DEPT) 极化转移增强distribution constant 分配系数diterpenoid 二萜diuresis 利尿，多尿diuretic 利尿剂diuretics 利尿剂，利尿药divinylbenzene 二乙烯苯dizziness and palptation 眩晕心悸dizziness and tinnitus 眩晕耳鸣doesahexaenoic acid 二十二碳六烯酸dopamine 多巴胺dotriacontane 三十二烷double lignans 双木脂素Double Quantum Filtered 1H-1H COSY (DQF-COSY) 双量子H-H 相关谱down-field 低磁场drain summer heat 解暑热drift 迁移drinking and urine 消渴dry dampness and transform phlegm 燥湿化痰Dryobalanops camphora龙脑香树Dryopteris crassirhizoma 贯众dual-modulation 双向调节ducitol 卫矛醇duct 导管duodenal tumours 十二指肠癌dysentery 痢疾dysmenorrheal 痛经dyspepsia 消化不良dyspnea and coughing 胸闷咳嗽Eechinacea purpurea紫花松果菊echinacea 紫锥菊eclampsia 子痫，惊厥eco-friendly 环境友好型的ecuelle 压榨法eczema 湿疹eduasarone 欧细辛醚eicosanoic acid 花生酸eicosapentaenoic acid 二十碳五烯酸electric field 电场electrodialysis 电渗析electrolysis 电泳electron donating 供电子electron impact 电子碰撞电离electron impact (EI) 电子轰击electronegativity 电负性electron-withdrawing 吸电子electrophilic 亲电子的，吸电子的electrophilic group 亲电基团electrophoresis 电泳electrospray ionization 电喷射电离electrospray ionization (ESI) 电喷雾电离elemane 榄香烷elevating 提升,提高eliminate dampness 排除湿气eliminating phlegm 排除痰液eliminated 消除的elimination 消除ellagitannins 逆没食子鞣质，鞣花酸鞣质eluate 洗脱液Embelia oblongifolia多脉酸藤子Embelia ribes 白花酸藤果emetine 吐根碱emmenagogue 调经药emodin 大黄素empirical 经验的emulgent 乳化剂emulsifier 乳化剂emulsin 苦杏仁酶emulsion乳汁/ 乳状液emulsion 苦杏仁苷酶enantiomer 对映体endocrine system 内分泌系统endoperoxide 内过氧化物endothelial 内皮的enfleurage 吸香法enol 烯醇enolic glycoside 烯醇苷enuresis 遗尿enzymatic 酶的enzymatic hydrolysis 酶水解enzymatic specificity 酶特异性enzyme 酶enzymolysis 酶解Ephedraceae 麻黄科Ephedrae Herba 麻黄ephedrine hydrochloride 盐酸麻黄碱ephedroxane 麻黄噁唑酮epiberberine 表小檗碱epicatechin 表儿茶素Epicatechin 表儿茶素epichlorohydrin 表氯醇epigastric腹上部的epimer 差向异构体epimeric structure 端基异构体epoxide ring 环氧环epoxide 环氧化物epoxy 环氧的epoxy 环氧基equatorial bond 平伏键equilibrium 平衡Eremostachys沙穗属ergosterol 麦角甾醇ergot 麦角，麦角菌Ericaceae 杜鹃花科Ericaceae 杜鹃花科eriocitrin 圣草次苷Erodii Herba 老鹳草erysipelas 丹毒erythrina species刺桐属esculentic acid 商陆酸esculentoside 商陆皂苷esculetin 七叶内酯，秦皮乙素esculin 七叶苷，秦皮甲素essential amino acids 必需氨基酸essential oil 精油ester 酯ester glycoside 酯苷ester linkage 酯键esterification 酯化ester saponins 酯皂苷estrogen 雌性激素Estrogen receptor 雌激素受体Estrogenic 雌激素的ethanol 乙醇ethereal oil 香精油，醚油（醚和杂醇油的混合物）etherification 醚化作用ethyl acetate 乙酸乙酯ethylene glycol 乙二醇eucalyptus oil 桉油eudesmane 桉烷eudesmol 桉醇，桉叶油醇eugenol 丁香酚eugenol type basil oil 丁香罗勒油euparotin 泽兰苦内酯Eupatorium rotundifolium圆叶泽兰Euphane 大戟烷Euphorbia大戟属Euphorbia fischeriana狼毒大戟Euphorbia kansui甘遂Euphorbia lathyris千金子Euphorbiaceae 大戟科eutectic point 共熔点evodiamine 吴茱萸碱excitatory有刺激性的，兴奋的excreting dampness 渗湿exhaustive 全面的，彻底的exocyclic 环外的expectorant 祛痰剂expelling 驱逐explosive 易爆炸的extinction coefficient 消光系数extract 提取物extraction 提取extremities sores 四肢疼痛exudate 渗出物F Fabaceae 豆科Faboideae 豆科蝶形花亚科Fagaceous plant 山毛榉科植物false negative 假阴性Fam. (familiar) 科fangchinoline 汉防己乙素，防己诺林碱faradiol 款冬二醇farnesane 金合欢烷farnesol 金合欢醇farnesyl pyrophosphate, FPP 焦磷酸金合欢酯fast atom bombardment (FAB) 快原子轰击fatty acid 脂肪酸fenchane 葑烷型fermentation 发酵fernane羊齿烷fernenol 羊齿烯醇Ferns 蕨类ferric chloride 氯化铁ferric trichloride 三氯化铁fertility 生育力，生育率fertilization 受精ferulic acid 阿魏酸feruoyl 阿魏酰基fever due to deficiency of Yin 阴虚发热fever from summer heat evil 暑邪发热fibrous root 须根Ficine 黄酮榕碱field desorption (FD) 场解吸法field desorption ionization 场解析电离field ionization 场致电离figwort root 玄参filariasis 丝虫病filter 过滤filtrate 滤液fire-purging drugs 清火药five watches 五更flammability 易燃，可燃性flavan-3,4-diol 黄烷-3,4-二醇Flavan-3,4-diols 3,4-二羟基黄烷醇flavan-3-ol 黄烷-3-醇Flavanol 黄烷醇Flavanones 黄烷酮类Flavanonols 二氢黄酮醇类Flavans 黄烷flavanyl 黄烷基Flavones 黄酮flavono-ellagitannins 黄酮类-鞣花酸鞣质flavonoid 黄酮flavonoid glycoside 黄酮苷Flavonoids 黄酮类Flavonols黄酮醇类Flavylium 花色基元flow chart 流程图fluid deficiency and constipation 津伤便秘fluorescence 荧光性foam 泡沫Foeniculum vulgare小茴香follicle hyperkeratosis 角化过度food accumulation and distending pain 食积胀痛formamide 甲酰胺formic acid 甲酸formidable 可怕的，令人畏惧的formononetin 刺芒柄花素Forsythia 连翘，连翘属植物Forsythia suspensa连翘forsythiaside 连翘酯苷forsythol 连翘酚fractionation 分馏法，分别fragment碎片fragmentation 碎片,破碎fragmentation mechanism 碎片裂解机制fragrance 芳香气味frankincense 乳香fraxetin 秦皮素fraxin 秦皮苷Fraxini Cortex 秦皮Fraxinus 白蜡树属fraxinus ornus花白腊木fraxinus rhynchophylla 苦枥白蜡树freckle 雀斑，斑点free fatty acid 游离脂肪酸free hydroxyl 游离羟基free radical 自由基free the network vessels and relieve pain 通络止痛friedelane木栓烷friedelin 木栓酮fritillaria tunbergii var. chekiangensis 东贝母frozen 凝固；冷冻fructan 果聚糖fructo-furanose unit 果糖呋喃糖单位fructose 果糖fructoside 果糖苷frufuranose 呋喃果糖fucose 夫糖, 岩藻糖fucoside 夫糖苷fucosterol 岩藻甾醇functional group 官能团fungus（fungi）真菌funnel 漏斗furan ring 呋喃环furanose 呋喃糖furanoside 呋喃糖苷furanspongin-3 呋喃海绵素-3furocoumarin 呋喃香豆素furostanol 呋甾烷醇fused 稠合fused-ring 稠环fused 乌本苷/G-毒毛花苷Ggalactomannan 半乳甘露聚糖galactose 半乳糖galactoside 半乳糖苷galactosyl 半乳糖基galacturonic acid 半乳糖醛酸galangin 三羟基黄酮gall aphid 五倍子蚜Galla Chinensis 五倍子gallate 没食子酸盐gallbladder 胆囊gallotannins 没食子鞣质galloyl esters 没食子酸酯galloylglucoses(GGs) 没食子酰基葡萄糖Calycosin-7-glucoside毛蕊异黄酮-7-葡糖苷ganoderma lucidum灵芝gas chromatography 气相色谱gas liquid chromatography 气液色谱gastric mucosa 胃粘膜gastrodia elata 天麻gastrodin 天麻苷gastroenteritis and dysentery 肠胃炎及痢疾gastroenteritis 胃肠炎gelidiaceae 石花菜科gelidium amansii 石花菜general debility with deficiency of vital essence 虚劳精亏genin 苷元genipin-1-O-gentiobioside 京尼平-1-O-龙胆双糖苷geniposide 京尼平苷Gentianaceae 龙胆科gentianine 龙胆碱Gentianose 龙胆三糖gentiobiose 龙胆双糖gentiopicroside,gentiopicrin 龙胆苦苷genus 属geometric 几何的geraniaceae 牻牛儿苗科geranial 香叶醛Geranii Herba 老鹳草geraniin 老鹳草素geraniol 香叶醇Geranium macrorrhizum大根老鹳草geranyl acetate 乙酸香叶酯geranyl pyrophosphate, GPP 焦磷酸香叶酯geranylfarnesyl pyrophosphate, GFPP 焦磷酸香叶基金合欢酯geranylgeranyl pyrophosphate, GGPP 焦磷酸香叶基香叶酯germacrane 吉马烷germacrone 吉马酮Gesneriaceae 苦苣苔科gigarginaceae杉藻科gigartina sps 杉藻gingkgolide 银杏内酯Ginkgetin 银杏素Ginkgo biloba 银杏ginkgo biloba 银杏科ginkgo folium (yinxingye) 银杏叶Ginkgoaceae 银杏科ginkgolide 银杏内酯ginkgolides 银杏内酯ginseng 人参ginsenoside 人参皂苷Ginseng Radix et Rhizoma 人参gitaloxigenin 吉他洛苷元gitoxigenin 羟基洋地黄毒苷元gitoxin 羟基洋地黄毒苷glacial 冰的，冰样的globular protein 球状蛋白。

专业英语词汇总结Section 1生药部分中药研究现状及中药现代化一、加强中国药用植物基础研究及其与中药现代化的联系/Strengthening basic researches on Chinese Medicinal Plants and its relations to realizing the modernization of CMM记载be recorded来源derived from中医药Traditional Chinese Medicine,short for TCM卫生事业health care,health undertakings中草药Chinese traditional medicinal herbs疗效reliable therapeutical effectstherapeutic[,θer?'pju:t?k]adj.治疗(学)的;疗法的；对身心健康有益的副作用side-effectsl中医药的健康理念和临床医疗模式体现了现代医学的发展趋势。

The health concept and clinical practice reflect the trend of modern science新的科学技术潮流（the new tide of science and technology）二、中药资源及其研究成果/Chinese Medicinal Plant resources and achievement of its scientific research中药资源(medicinal plant resources)普查(surveys)专项研究(special projects)药用植物资源(the Chinese medicinal resources)科学鉴定(scientific identification)化学成分(chemical constituents)药理实验(pharmacological experiments临床适应症(clinical applications)研究(projects)新著作(new works)各论(monographs)手册(manuals)《中国药典》The pharmacopoeia of the people’s Republic of China药典Pharmacopoeia药用植物学Pharmaceutical Botany本草学Herbology中药学The Chinese Materia Medica药用植物分类学Pharmaceutical Plant Taxonomy植物化学Phytochemistry植物化学分类学Plant Chemotaxonomy药用植物志Flora of Medicinal Plant中药药剂学traditional Chinese Pharmaceutics中药炮制学Science of processing Chinese Crude Drugs中药鉴定学Identification of Traditional Chinese Medicine中药药理学Pharmacology of Traditional Chinese Medicines青蒿素artemisin奎宁quinine、氯奎宁chloroquine衍生物derivatives氯奎宁耐受性疟疾chloroquine resistant malaria急性疟疾pernicious malaria脑部疟疾cerebral malaria显著疗效marked effect chloroquine resistant malaria/抗氯喹啉疟疾Pernicious（有害的）malaria/急性疟疾cerebral malaria/脑疟疾derivatives/衍生物quinine/喹啉含有氮原子的化合物，在英文命名中多以-ine结尾Mono-/一Di-/二Tri-/三Tetra-/四Petan-/五Hexa-/六Hepta-/七Octa-/八Nona-/九Deca-/十三尖杉酯碱harringtonine、高三尖杉酯碱homoharringtonine白血病leukemia和恶性淋巴瘤malignant lymphoma银杏黄酮ginkgetin丹参酮tanshinon IIA治疗冠心病coronary heart diseasesNew drug developments/新药开发Health products/保健品质量控制Quality control修订revise常用中药common-used Chinese materia medica国家标准the national standards三、中药所面临的挑战/Chinese Medicinal Herbs Facing a Challenge中成药及其制剂traditional Chinese patent medicines and preparations基础研究basic researches生产production、流通marketing研究researchIdentification of species/品种鉴定鉴定和鉴别identifying and clarifying变种varieties伪品false matters。

双语生物化学词汇Glossary of Biochemistry BilinguallyAAbsolute configuration（绝对构型）The configuration of four different substituent groups around an asymmetric carbon atom, in relation to u- and i.-glyceraldehyde. Absorption（吸收）: transport of the products of digestion from the intestinal tract into the blood.Acceptor control（受体控制）:The regulation of the rate of respiration by the availability of ADP as phosphate group acceptor.Accessory pigments（辅助色素）:Visible light-absorbing pigments (carotenoids, xanthophyll, and phycobilins藻胆素) in plants and photosynthetic bacteria that complement chlorophylls in trapping energy from sunlight.Acidosis（酸中毒）: A metabolic condition in which the capacity of the body to buffer is diminished; usually accompanied by decreased blood pH.Actin（肌动蛋白）: A protein making up the thin filaments（细丝）of muscle; also an important component of the cytoskeleton of many eukaryotic cells.Activation energy(ΔG*)（活化能）: The amount of energy (in joules) required to convert all the molecules in 1 mole of a reacting substance from the ground state to the transition state.Activator:（活化物、激活剂）(1) A DNA-binding protein that positively regulates the expression of one or more genes; that is, transcription rates increase when an activator is bound to the DNA. (2) A positive modulator of an allosteric enzyme.Active site:（活性部位）The region of an enzyme surface that binds the substrate molecule and catalytically transforms it; also known as the catalytic site.Active transport:（主动运输）Energy-requiring transport of a solute across a membrane in the direction of increasing concentration.Activity:（活度）The true thermodynamic activity or potential of a substance, as distinct from its molar concentration.Activity coefficient:（活度系数）The factor by which the numerical value of the concentration of a solute must be multiplied to give its true thermodynamic activity. Adenosine 3',5'-cyclic monophosphate: See cyclic AMP.Adenosine diphosphate: See ADP.Adenosine triphosphate: See ATP.Adipocyte:（脂肪细胞）An animal cell specialized for the storage of fats (triacylglycerols).Adipose tissue:（脂肪组织）Connective tissue specialized for the storage of large amounts of triacylglycerols.ADP (adenosine diphosphate):A ribonucleoside diphosphate serving as phosphate group acceptor in the cell energy cycle.Aerobe:（需氧生物）An organism that lives in air and uses oxygen as the terminal electron acceptor in respiration.Aerobic: Requiring or occurring in the presence of oxygen.Alcohol fermentation:（乙醇发酵）The anaerobic conversion of glucose to ethanol via glycolysis. See also fermentation.Aldose:（醛糖）A simple sugar in which the carbonyl carbon atom is an aldehyde; that is, the carbonyl carbon is at one end of the carbon chain.Alkalosis:（碱中毒）A metabolic condition in which the capacity of the body to buffer is diminished; usually accompanied by an increase in blood pH.Allosteric enzyme:（变/别构效应） A regulatory enzyme, with catalytic activity modulated by the noncovalent binding of a specific metabolite at a site other than the active site.Allosteric protein: （变/别构蛋白）A protein (generally with multiple subunits) with multiple ligand-binding sites, such that ligand binding at one site affects ligand binding at another.Allosteric site: （变/别构部位）The specific site on the surface of an allosteric enzyme molecule to which the modulator or effector molecule is bound.α helix:（α-螺旋）A helical conformation of a polypeptide chain, usually right-handed, with maximal intrachain hydrogen bonding; one of the most common secondary structures in proteins.Ames test:A simple bacterial test for carcinogens, based on the assumption that carcinogens are mutagens.Amino acid activation:（氨基酸活化）ATP-dependent enzymatic esterification of the carboxyl group of an amino acid to the 3'-hydroxyl group of its corresponding tRNA. Amino acids:（氨基酸）an Amino-substituted carboxylic acids, the building blocks of proteins.Amino-terminal residue:（氨基末端残基）The only amino acid residue in a polypeptide chain with a free a-amino group; defines the amino terminus of the polypeptide.Aminoacyl-tRNA:（氨酰tRNA）An aminoacyl ester of a tRNA.Aminoacyl-tRNA synthetases:（氨酰tRNA合成酶）Enzymes that catalyze synthesis of an aminoacyl-tRNA at the expense of ATP energy.Aminotransferases:（氨基转移酶）Enzymes that catalyze the transfer of amino groups fromα-amino to α-keto acids; also called transaminases.Ammonotelic:（排氨的）Excreting excess nitrogen in the form of ammonia. Amphibolic pathway:（双向代谢途径）A metabolic pathway used in both catabolism and anabolism.Amphipathic:（双亲的）Containing both polar and nonpolar domains. Ampholyte:（两性电解质）A substance that can act as either a base or an acid. Amphoteric:（两性的）Capable of donating and accepting protons, thus able to serve as an acid or a base.Anabolisim:（合成代谢）The phase of intermediary metabolism concerned with the energy-requiring biosynthesis of cell components from smaller precursors. Anaerobe:（厌氧生物）An organism that lives without oxygen. Obligate anaerobes （专性厌氧生物）die when exposed to oxygen.Anaerobic:（厌氧的）Occurring in the absence of air or oxygen.Anaplerotic reaction:（回补反应）An enzyme-catalyzed reaction that can replenish the supply of intermediates in the citric acid cycle.A ngstrom (Ǻ):（唉）A unit of length (10-8cm) used to indicate molecular dimensions. Anhydride:（酸酐）The product, of the condensation of two carboxyl or phosphate groups in which the elements of water are eliminated to form a compound with the general structure R—X—0—X—R, where X is either carbon or phosphorus.Anion-exchange resin:（阴离子交换树脂）A polymeric resin with fixed cationic groups; used in the chromatographic separation of anions.Anomers:（异头物、端基异构体）Two stereoisomers of a given sugar that differ only in the configuration about the carbonyl (anomeric) carbon atom.Antibiotic:（抗生素）One of many different organic compounds that are formed and secreted by various species of microorganisms and plants, are toxic to other species, and presumably have a defensive function.Antibody:（抗体）A defense protein synthesized by the immune system of vertebrates. See also immunoglobulin.Anticodon:（反密码子） A specific sequence of three nucleotides in a tRNA, complementary to a codon for an amino acid in an mRNA.Antigen:（抗原）A molecule capable of eliciting the synthesis of a specific antibody in vertebrates.Antiparallel:（反平行）Describing two linear polymers that are opposite in polarity or orientation.Antiport:（反向转运）Cotransport of two solutes across a membrane in opposite directions.Apoenzyme:（酶蛋白）The protein portion of an enzyme, exclusive of any organic or inorganic cofactors or prosthetic groups that might be required for catalytic activity. Apolipoprotein:（脱辅基脂蛋白）The protein component of a lipoprotein. Apoprotein: （脱辅基蛋白）The protein portion of a protein, exclusive of any organic or inorganic cofactors or prosthetic groups that might be required for activity. Apoptosis:（细胞凋亡）(app'-a-toe'-sis) Programmed cell death, in which a cell bringsabout its own death and lysis, signaled from outside or programmed in its genes, by systematically degrading its own macromolecules.Arrestin:（抑制蛋白） A family of proteins that bind to the phosphorylated carboxyl-terminal region of serpentine receptors, preventing their interactions with G proteins and thereby terminating the signal through those receptors.Asymmetric carbon atom:（不对称碳原子）A carbon atom that is covalently bonded to four different groups and thus may exist in two different tetrahedral configurations. ATP (adenosine triphosphate): A ribonucleoside 5'-triphosphate functioning as a phosphate group donor in the cell energy cycle; carries chemical energy between metabolic pathways by serving as a shared intermediate coupling endergonic and exergonic reactions.ATP synthase:（ATP合酶）An enzyme complex that forms ATP from ADP and phosphate during oxidative phosphorylation in the inner mitochondrial membrane or the bacterial plasma membrane, and during photophosphorylation in chloroplasts. ATPase:（ATP酶）An enzyme that hydrolyzes ATP to yield ADP and phosphate; usually coupled to some process requiring energy.Attenuator:（弱化子）An RNA sequence involved in regulating the expression of certain genes; functions as a transcription terminator.Autotroph:（自养生物）An organism that can synthesize its own complex molecules from very simple carbon and nitrogen sources, such as carbon dioxide and ammonia. Auxin:（植物生长素）A plant growth hormone.Auxotrophic mutant (auxotroph):（营养缺陷突变体）A mutant organism defective in the synthesis of a given biomolecule, which must therefore be supplied for the organism's growth.Avogadro's number: The number of molecules in a gram molecular weight (a mole) of any compound (6.02 × 1023).BBack-mutation:（回复突变）A mutation that causes a mutant gene to regain its wild-type base sequence.Bacteriophage (phage):（噬菌体）A virus capable of replicating in a bacterial cell. Basal metabolic rate:（基础代谢率）The rate of oxygen consumption by an animal's body at complete rest, long after a meal.Base pair:（碱基对）Two nucleotides in nucleic acid chains that are paired by hydrogen bonding of their bases; for example, A with T or U, and G with C.β conformation:（β构象）、An extended, zigzag arrangement of a polypeptide chain; a common secondary structure in proteins.βoxidation:（β氧化）Oxidative degradation of fatty acids into acetyl-CoA by successive oxidations at the β-carbon atom.β-turn:（β转角）A type of secondary structure in polypeptides consisting of four aminoacid residues arranged in a tight turn so that the polypeptide turns back on itself. Bilayer:（双分子层）A double layer of oriented amphipathic lipid molecules, forming the basic structure of biological membranes. The hydrocarbon tails face inward to form a continuous nonpolar phase.Bile salts:（胆酸盐）Amphipathic steroid derivatives with detergent properties, participating in digestion and absorption of lipids.Binding energy:（吸附能）The energy derived from noncovalent interactions between enzyme and substrate or receptor and ligand.Binding site:（结合部位）The crevice or pocket on a protein in which a ligand binds. Biocytin:（生物胞素）The conjugate amino acid residue arising from covalent attachment of biotin, through an amide linkage, to a Lys residue.Biomolecule:（生物分子）An organic compound normally present as an essential component of living organisms.Biopterin:（生物喋呤）An enzymatic cofactor derived from pterin and involved in certain oxidation-reduction reactions.Biosphere:（生物圈）All the living matter on or in the earth, the seas, and the atmosphere.Biotin:（生物素）A vitamin; an enzymatic cofactor involved in carboxylation reactions. Bond energy:（键能）The energy required to break a bond.Branch migration:（分支迁移）Movement of the branch point in branched DNA formed from two DNA molecules with identical sequences. See also Holliday intermediate.Buffer:（缓冲液）A system capable of resisting changes in pH, consisting of a conjugate acid-base pair in which the ratio of proton acceptor to proton donor is near unity.CCalorie:（卡）The amount of heat required to raise the temperature of 1.0 g of water from 14.5 to 15.5 °C. One calorie (cal) equals 4.18 joules (J).Calvin cycle:（Calvin循环）The cyclic pathway used by plants to fix carbon dioxide and produce triose phosphates.cAMP:See cyclic AMP.cAMP receptor protein (CRP): （cAMP受体蛋白）A specific regulatory protein that controls initiation of transcription of the genes producing the enzymes required for a bacterial cell to use some other nutrient when glucose is lacking. Also called catabolite gene activator protein (CAP)，降解物基因活化蛋白.CAP:See catabolite gene activator protein.Capsid:（衣壳）The protein coat of a virion or virus particle.Carbanion:（碳负离子）A negatively charged carbon atom.Carbocation: （碳正离子）A positively charged carbon atom; also called a carboniumion.Carbon-assimilation reactions:（碳同化反应）Reaction sequences in which atmospheric CO2 is converted into organic compounds.Carbon-fixation reaction:（固碳反应）The reaction catalyzed by rubisco during photosynthesis, or by other carboxylases, in which atmospheric CO2is initially incorporated into an organic compound.Carboxyl-terminal residue:（羧基末端残基）The only amino acid residue in a polypeptide chain with a free a-carboxyl group; defines the carboxyl terminus of the polypeptide.Carotenoids:（类葫罗卜素）Lipid-soluble photosynthetic pigments made up of isoprene units.Catabolism:（分解代谢）The phase of intermediary metabolism concerned with the energy-yielding degradation of nutrient molecules.Catabolite gene activator protein (CAP):See cAMP receptor protein.Catalytic site:（催化部位）See active site.Catecholamines:（儿茶酚胺类）Hormones, such as epinephrine, that are amino derivatives of catechol.Catenane:（连环体）Circular polymeric molecules with a noncovalent topological link resembling the links of a chain.Cation-exchange resin:（阳离子交换树脂）An insoluble polymer with fixed negative charges; used in the chromatographic separation of cationic substances.cDNA: See complementary DNA.Central dogma:（中心法则）The organizing principle of molecular biology: genetic information flows from DNA to RNA to protein.Centromere:（着丝粒） A specialized site within a chromosome, serving as the attachment point for the mitotic or meiotic spindle.Cerebroside(脑苷酯) Sphingolipid containing one sugar residue as a head group. Channeling:（生物合成途径限制作用)The direct transfer of a reaction product (common intermediate) from the active site of one enzyme to the active site of a different enzyme catalyzing the next step in a sequential pathway.Chemiosmotic coupling:（化学渗透偶联）Coupling of ATP synthesis to electron transfer via an electrochemical H+ gradient across a membrane.Chemotaxis（向化性）:A cell's sensing of and movement toward, or away from, a specific chemical agent.Chemotroph:（化能生物）An organism that obtains energy by metabolizing organic compounds derived from other organisms.Chiral center:（手性中心）An atom with substituents arranged so that the molecule is not superimposable on its mirror image.Chiral compound:（手性化合物）A compound that contains an asymmetric center(chiral atom or chiral center) and thus can occur in two nonsuperimposable mirror-image forms (enantiomers).Chlorophylls:（叶绿素）A family of green pigments functioning as receptors of light energy in photosynthesis; magnesium-porphyrin complexes.Chloroplasts:（叶绿体）Chlorophyll-containing photosynthetic organelles in some eukaryotic cells.Chromatin:（染色质）A filamentous complex of DNA, histones, and other proteins, constituting the eukaryotic chromosome.Chromatography:（层析）A process in which complex mixtures of molecules are separated by many repeated partitionings between a flowing (mobile) phase and a stationary phase.Chromosome:（染色体）A single large DNA molecule and its associated proteins, containing many genes; stores and transmits genetic information.Chylomicron:（乳糜微粒）A plasma lipoprotein consisting of a large droplet of triacylglycerols stabilized by a coat of protein and phospholipid; carries lipids from the intestine to the tissues.cis and trans isomers:（顺反异构体）See geometric isomers.Cistron:（顺反子）A unit of DNA or RNA corresponding to one gene.Citric acid cycle:（柠檬酸循环）A cyclic system of enzymatic. reactions for the oxidation of acetyl residues to carbon dioxide, in which formation of citrate is the first step; also known as the Krebs cycle or tricarboxylic acid cycle.Clones:（克隆）The descendants of a single cell.Cloning:The production of large numbers of identical DNA molecules, cells, or organisms, from a single ancestral DNA molecule, cell, or organism.Closed system:（封闭系统）A system that exchanges neither matter nor energy with the surroundings. See also system.Cobalamin:（钴胺素）See cocnzyme B12.Codon:（密码子）A sequence of three adjacent nucleotides in a nucleic acid that codes for a specific amino acid.Coenzyme:（辅酶）An organic cofactor required for the action of certain enzymes; often contains a vitamin as a component.Coenzyme A: （辅酶A）A pantothenic acid-containing coenzyme serving as an acyl group carrier in certain enzymatic reactions.Coenzyme B12: An enzymatic cofactor derived from the vitamin cobalamin, involved in certain types of carbon skeletal rearrangements.Cofactor(辅助因子) An inorganic ion or a coenzyme required for enzyme activity. Cognate:（相关的）Describing two biomolecules that normally interact; for example, an enzyme and its normal substrate, or a receptor and its normal ligand.Cohesive ends:（粘性末端）See sticky ends.Cointegrate:（共整合）An intermediate in the migration of certain DNA transposons in which the donor DNA and target DNA are covalently attached.Colligative properties:（依数性）Properties of solutions that depend on the number of solute particles per unit volume; for example, freezing-point depression.Common intermediate:（共同中间产物）A chemical compound common to two chemical reactions, as a product of one and a reactant in the other.Competitive inhibition:（竞争性抑制作用）A type of enzyme inhibition reversed by increasing the substrate concentration; a competitive inhibitor generally competes with the normal substrate or ligand for a protein's binding site.Complementary:（互补）Having a molecular surface with chemical groups arranged to interact specifically with chemical groups on another molecule.Complementary DNA (cDNA): A DNA used in DNA cloning, usually made by reverse transcriptase; complementary to a given mRNA.Configuration:（构型）The spatial arrangement of an organic molecule that is conferred by the presence of either (1) double bonds, about which there is no freedom of rotation, or (2) chiral centers, around which substituent groups are arranged in a specific sequence. Configurational isomers cannot be interconverted without breaking one or more covalent bonds.Conformation:（构象）The spatial arrangement, of substituent groups that are free to assume different positions in space, without breaking any bonds, because of the freedom of bond rotation.Conjugate acid-base pair:（共扼酸碱对） A proton donor and its corresponding deprotonated species; for example, acetic acid (donor) and acetate (acceptor). Conjugate redox pair: （共扼氧还对）An electron donor and its corresponding electron acceptor form; for example, Cu+ (donor) and Cu2+ (acceptor), or NADH (donor) and NAD+ (acceptor).Conjugated protein:（结合蛋白质）A protein containing one or more prosthetic groups.Consensus sequence:（一致序列）A DNA or amino acid sequence consisting of the residues that occur most commonly at each position within a set of similar sequences. Conservative substitution:（保守性置换）Replacement of an amino acid residue in a polypeptide by another residue with similar properties; for example, substitution of Glu by Asp.Constitutive enzymes:（组成酶）Enzymes required at all times by a cell and present at some constant level; for example, many enzymes of the central metabolic pathways. Sometimes called house-keeping enzymes.Contour length (外形长度): The length of a helical polymeric molecule as measured along the molecule's helical axis.Corticosteroids（皮质类固醇激素）Steroid hormones formed by the adrenal cortex. Cotransport:（共转运）The simultaneous transport, by a single transporter, of twosolutes across a membrane. See antiport, symport.Coupled reactions:（偶联反应）Two chemical reactions that have a common intermediate and thus a means of energy transfer from one to the other.Covalent bond:（共价键）A chemical bond that involves sharing of electron pairs. Cristae:（嵴）Infoldings of the inner mitochondrial membrane.CRP（cAMP受体蛋白）See cAMP receptor protein.Cyclic AMP (cAMP):A second messenger within cells; its formation by adenylyl cyclase is stimulated by certain hormones or other molecular signals.Cyclic electron flow:（循环电子流）In chloroplasts, the light-induced flow of electrons originating from and returning to photosystem I.Cyclic photophosphorylation: （循环光合磷酸化）ATP synthesis driven by cyclic electron flow through photosystem I.Cyclin:（细胞周期蛋白）One of a family of proteins that activate cyclin-dependent protein kinases and thereby regulate the cell cycle.Cytochromes:（细胞色素）Heme proteins serving as electron carriers in respiration, photosynthesis, and other oxidation-reduction reactions.Cytokine:（细胞因子）One of a family of small secreted proteins (such as interleukins or interferons) that activate cell division or differentiation by binding to plasma membrane receptors in sensitive cells.Cytokinesis:（胞质分裂）The final separation of daughter cells following mitosis. Cytoplasm:（细胞质）The portion of a cell's contents outside the nucleus but within the plasma membrane; includes organelles such as mitochondria.Cytoskeleton:（细胞骨架）The filamentous network providing structure and organization to the cytoplasm; includes actin filaments, microtubules, and intermediate filaments.Cytosol:（细胞浆）The continuous aqueous phase of the cytoplasm, with its dissolved solutes; excludes the organelles such as mitochondria.DDalton:（道尔顿）The weight of a single hydrogen atom (1.66 x I0-24 g).Dark reactions:（暗反应）See carbon-assimilation reactions.De novo pathway:（从头合成）Pathway for synthesis of a biomolecule, such as a nucleotide, from simple precursors; as distinct from a salvage pathway. Deamination:（脱氨基作用）The enzymatic removal of amino groups from biomolecules such as amino acids or nucleotides.Degenerate code:（兼并密码）A code in which a single element in one language is specified by more than one element in a second language.Dehydrogenases:（脱氢酶类）Enzymes catalyzing the removal of pairs of hydrogen atoms from their substrates.Deletion mutation:（删除突变）A mutation resulting from the deletion of one or morenucleotides from a gene or chromosome.Denaturation:（变性）Partial or complete unfolding of the specific native conformation of a polypeptide chain, protein, or nucleic acid.Denatured protein:（变性蛋白）A protein that has lost its native conformation by exposure to a destabilizing agent such as heat or detergent.Deoxyribonucleic acid; See DNA.Deoxyribonucleotides:（脱氧核糖核苷酸）Nucleotides containing 2-deoxyribose as the pentose component.Desaturases:（去饱和酶）Enzymes that catalyze the introduction of double bonds into the hydrocarbon portion of fatty acids.Desolvation:（脱水）In aqueous solution, the release of bound water surrounding a solute.Dextrorotatory isomer:9右旋异构体） A stercoisomer that rotates the plane of plane-polarized light clockwise.Diabetes mellitus:（糖尿病）A metabolic disease resulting from insulin deficiency; characterized by a failure in glucose transport from the blood into cells at normal glucose concentrations.Dialysis:（透析）Removal of small molecules from a solution of a macromolecule, by allowing them to diffuse through a semipermeable membrane into water.Differential centrifugation:（差速离心）Separation of cell organelles or other particles of different size by their different rates of sedimentation in a centrifugal field. Differentiation:（分化）Specialization of cell structure and function during embryonic growth and development.Diffusion:（扩散）The net movement, of molecules in the direction of lower concentration.Digestion:（消化）Enzymatic hydrolysis of major nutrients in the gastrointestinal system to yield their simpler components.Diploid:（二倍体）Having two sets of genetic information; describing a cell with two chromosomes of each type.Dipole;（双极分子）A molecule having both positive and negative charges.Diprotic acid: An acid having two dissociable protons.Disaccharide:（二糖） A carbohydrate consisting of two covalently joined monosaccharide units.Dissociation constant:（解离常数）(1) An equilibrium constant (K d)for the dissociation of a complex of two or more biomolecules into its components; for example, dissociation of a substrate from an enzyme. (2) The dissociation constant (Ka) of an acid, describing its dissociation into its conjugate base and a proton.Disulfide bridge:(二硫桥)A covalent cross link between two polypeptide chains formed by a cystine residue (two Cys residues).DNA (deoxyribonucleic acid)： A polynucleotide having a specific sequence of deoxyribonucleotide units covalently joined through 3', 5'-phosphodiester bonds; serves as the carrier of genetic information.DNA chimera:（DNA嵌合）A DNA containing genetic information derived from two different species.DNA cloning:Sec cloning.DNA library:（DNA文库）A collection of cloned DNA fragments.DNA ligase：（DNA连接酶）An enzyme that creates a phosphodiester bond between the 3' end of one DNA segment, and the 5' end of another.DNA looping:（DNA出环）The interaction of proteins bound at distant sites on a DNA molecule so that the intervening DNA forms a loop.DNA microarray:（DNA微阵列）A collection of DNA sequences immobilized on a solid surface, with individual sequences laid out in patterned arrays that can be probed by hybridization.DNA polymerase:（DNA聚合酶）An enzyme that catalyzes template-dependent synthesis of DNA from its deoxyribonucleoside 5'-triphosphate precursors.DNA replicase system：（DNA复制酶系统）The entire complex of enzymeH and specialized proteins required in biological DNA replication.DNA supercoiling:（DNA超螺旋化）The coiling of DNA upon itself, generally as a result of bending, underwinding, or overwinding of the DNA helix.DNA transposition:（DNA转座）See transposition.domain:（结构域）A distinct structural unit of a polypeptide; domains may have separate functions and may fold as independent, compact units.Double helix:（双螺旋）The natural coiled conformation of two complementary, antiparallel DNA chains.Double-reciprocal plot:（双倒数作图）A plot, of 1/Vo versus 1/[S], which allows a more accurate determination of Vmax and Km than a plot of V versus [S]; also called the Lineweaver-Burk plot,EE'°: 标准还原电位See standard reduction potential.E. coli (Escherichia coli):（大肠杆菌）A common bacterium found in the small intestine of vertebrates; the most well-studied organism.Electrochemical gradient:（电化学梯度）The sum of the gradients of concentration and of electric charge of an ion across a membrane; the driving force for oxidative phosphorylation and photophosphorylation.Electrochemical potential: （电化学势）The energy required to maintain a separation of charge and of concentration across a membrane.Electrogenic:（生电的）Contributing to an electrical potential across a membrane.Electron acceptor:（电子受体） A substance that receives electrons in an oxidation-reduction reaction.Electron carrier:（电子载体）A protein, such as a flavoprotein or a cytochrome, that can reversibly gain and lose electrons; functions in the transfer of electrons from organic nutrients to oxygen or some other terminal acceptor.Electron donor:（电子供体） A substance that donates electrons in an oxidation-reduction reaction.Electron transfer:（电子转移）Movement of electrons from substrates to oxygen via the carriers of the respiratory (electron transfer) chain.Electrophile:（亲电剂）An electron-deficient group with a strong tendency to accept electrons from an electron-rich group (nucleophile).Electrophoresis（电泳）: Movement of charged solutes in response to an electrical field; often used to separate mixtures of ions, proteins, or nucleic acids.Electroporation:（电穿孔法）Introduction of macromolecules into cells after rendering the cells transiently permeable by the application of a high-voltage pulse.Elongation factors:（延长因子）Specific proteins required in the elongation of polypeptide chains by ribosomes.Eluate:（流出液）The effluent from a chromatographic column.Enantiomers:（对映异构体）Stereoisomers that are nonsuperimposable mirror images of each other.End-product inhibition:See feedback inhibition.Endergonic reaction(耗能反应): A chemical reaction that consumes energy (that is, for which ΔG is positive).Endocrine glands:（内分泌腺）Groups of cells specialized to synthesize hormones and secrete them into the blood to regulate other types of cells.Endocytosis:（内吞体）The uptake of extracellular material by its inclusion within a vesicle formed by an invagination of the plasma membrane.Endonuclease:（内切核酸酶）An enzyme that hydrolyzes the interior phosphodiester bonds of a nucleic acid; that is, it acts at points other than the terminal bonds. Endoplasmic reticulum:（内质网）An extensive system of double membranes in the cytoplasm of eukaryotic cells; it encloses secretory channels and is often studded with ribosomes (rough endoplasmic reticulum).Endothermic reaction:（吸热反应）A chemical reaction that takes up heat (that is, for which ΔH is positive).Energy charge:（能荷）The fractional degree to which the ATP/ADP/AMP system is filled with high-energy phosphate groups.Energy coupling:（能量偶联）The transfer of energy from one process to anotlier. Enhancers:（增强子）DNA sequences that facilitate the expression of a given gene;。

Mild,efficient and rapid O-debenzylation of ortho -substituted phenols with trifluoroacetic acidSteven Fletcher *,Patrick T.Gunning *Department of Chemistry,University of Toronto,Mississauga,ON L5L 1C6,Canadaa r t i c l e i n f o Article history:Received 21May 2008Revised 2June 2008Accepted 4June 2008Available online 10June 2008a b s t r a c tThe mild and efficient deblocking of aryl benzyl ethers with TFA is reported.Cleavage was fastest with ortho -electron-withdrawing groups on the phenolic ring,which we have attributed to a proton chelation effect,furnishing the deprotected phenols in excellent yields.The corresponding para -methoxybenzyl,allyl and iso -propyl ethers were also cleanly removed under these conditions.In addition,the selective aryl benzyl ether debenzylation in the presence of benzyl ester,Cbz carbamate and Boc carbamate func-tionalities was also observed.Crown Copyright Ó2008Published by Elsevier Ltd.All rights reserved.Phosphotyrosines feature in the design of inhibitors of several protein targets,including protein tyrosine phosphatase 1B (PTP1B).1However,these moieties suffer from hydrolytic lability to cellular phosphatases and poor cell penetration due to the asso-ciated dianionic charge.1To address these issues,salicylic acid derivatives (and closely-related analogues)have become popular mimetics of phosphotyrosine in small molecule inhibitors.1–5Turk-son et al.have recently reported on NSC74859(1),a potent,sali-cylic acid-based inhibitor of the oncogenic protein Stat3.6As part of our structure–activity relationship (SAR)studies on NSC74859(1),we sought to debenzylate both the phenol ether and benzoate ester in 2without reducing the aryl-bromide bond,a common undesired side reaction that occurs with hydrogen gas and Pd/C catalyst.7O -Benzyl-protected phenols are known to undergo debenzyla-tion with trifluoroacetic acid (TFA)8by an initial protonation of the weakly basic phenol oxygen,although additives such as strongorganic acids (e.g.,trifluoromethanesulfonic acid 9)or a large excess of nucleophilic scavenger (e.g.,thioanisole,which accelerates the reaction by a ‘push–pull’mechanism 10)are typically required.Re-cent work by Ploypradith et al.describes the mild deprotection of aromatic ethers with sub-stoichiometric para -toluenesulfonic acid on solid support.11In a special case,O -benzyl-protected ortho -nitrophenol was cleaved rapidly (<5min)with neat TFA,12which we considered was due to the ability of the substrate to chelate a proton since the structurally-similar ortho -hydroxybenzoates (salicylates)are well-known to chelate copper ions and iron ions.We reasoned that 2(and indeed 3)may similarly undergo acceler-ated debenzylation with TFA.In fact,as shown in Scheme 1,treat-ment of 2(or 3)with a 1:1mixture of TFA/toluene led to rapid debenzylation (5min for 2;1h for 3)in 91%yield for 2(or 85%yield for 3).In this Letter,we will explore the structural require-ments of the phenol component that increase the lability of the O -benzyl phenol ether bond in the presence of TFA.In addition,0040-4039/$-see front matter Crown Copyright Ó2008Published by Elsevier Ltd.All rights reserved.*Tel.:+19058285354;fax:+19058285425(P.T.G.).E-mail addresses:steven.fletcher@utoronto.ca (S.Fletcher),patrick.gunning@utoronto.ca (P.T.Gunning).Tetrahedron Letters 49(2008)4817–4819Contents lists available at ScienceDirectTetrahedron Lettersj o ur na l h om e pa ge :w w w.e ls e v ie r.c o m/lo c at e/t et l e twe will explore the selectivity of this mild debenzylation tech-nique with respect to other aromatic ethers and examine the sta-bility of other benzyl-based protecting groups to these reaction conditions.A series of 12O -benzyl-protected phenols was prepared by standard procedures in near quantitative yields.Each of these ethers was then deprotected with a 1:1mixture of TFA/toluene;our observations are summarized in Table 1.In certain cases,O ?C benzyl migration (Friedel–Crafts reaction)by-products (610%)were occasionally inseparable from the product by silica gel flash column chromatography.Thus,several benzyl cation cap-tors were investigated for their abilities to improve yields and puri-ties of the debenzylation reactions.Three to ten equivalents of p -cresol,anisole and triethylsilane were employed,but these exerted little effects on reducing by-product formation.Conversely,we dis-covered that including the more nucleophilic scavenger thioanisole as an additive to the co-solvent toluene typically,after silica gel flash column chromatography,furnished products in P 95%puri-ties (and higher yields),as judged by 1H NMR.Nevertheless,we envisaged any Friedel–Crafts impurities would be more readily separable on slightly more complex aryl benzyl ethers,as we ob-served with the substrates shown in Scheme 1and Tables 3and 4(>99%purities (1H NMR)in each case).Whilst likely leading to even higher yields and purities,large excesses of thioanisole (50equiv)are also known to accelerate TFA-mediated debenzyla-tion.10However,in our hands just 3equiv of thioanisole had little effect on the rate of debenzylation,allowing us to attribute the deprotection rates solely to the structure of the phenol.Electron-rich phenols are good scavengers of benzyl cations,13and since preliminary experiments with electron-rich phenols generated complex mixtures of Friedel–Crafts by-products under these deb-enzylation conditions,we chose to investigate only electron-poor phenols in this study.O -Benzyl-protected phenols with p -ortho -electron-withdraw-ing groups (6a ,6b ,6d ,6f )were swiftly (several in less than 3h cf.24h for unsubstituted phenol 6l )and cleanly debenzylated,with less than 5%of the undesired C-benzylated phenol by-prod-ucts.In contrast,meta -and para -electron-withdrawing groups slo-wed down the debenzylation (e.g.,entries 6g and 6h ),relative to the control compound 6l ,which itself could only be obtained in moderate purity by this method.The r -withdrawing (and p -donating)bromophenols 6i –k were insufficiently deactivated to benzyl cation scavenging and were contaminated with several by-products.Importantly,n -butyl benzyl ether 8was unaffected by TFA under the reaction conditions,indicating this procedure is selective for aryl benzyl ethers.In addition,the results in Table 1suggest that this procedure is suitable only for phenols substituted with p -electron-withdrawing groups.Since the debenzylation mechanism with TFA proceeds via an initial protonation of the phenol ether oxygen,the more available the ether oxygen lone pairs are,the faster the reaction will be.Hence,the slower reaction times for the phenols bearing meta -and para -electron-withdrawing groups make sense,although this is not true for the ortho -functionalized aryl benzyl ethers.As hypothesized for the bis-benzyl salicylate derivative 2earlier,we considered these ortho -substituted phenols were capable of chelat-ing the acidic hydrogen atom from TFA which therein facilitated the acid-mediated debenzylation via a six-membered cyclic inter-mediate,as proposed in Scheme 2.A similar chelation intermediate has been put forward by Baldwin and Haraldsson to account for the Lewis acid MgBr 2-mediated debenzylation of aromatic benzyl ethers ortho to an aldehyde group.14Accordingly,to test this hypothesis we expanded this series of ortho -substituted aryl benzyl ethers,and the results from their deb-enzylation reactions with TFA are summarized in Table 2.These substrates have been listed in order of increasing approximateTable 1TFA-mediated debenzylation of O -benzyl-protected phenols aTFAtolueneOBnROHR67Substrate RTime (h)b Yield c (%)6a o -CO 2Me,m d -NHAc 5min 936b o -CO 2Me 5min 946c p -CO 2Me 36e 63(85f )6d o -CO 2Bn 5min 936e p -CO 2Bn 36e 58(79f )6f o -NO 23976g m -NO 236e 75(98f )6h p -NO 236e 66(98f )6i o -Br 16—g 6j m -Br 30—g 6k p -Br 36—g 6lH 24—gn -BuOBn (8)—24No reactionaThe reaction was carried out with 6(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt,with 3equiv of thioanisole.bTime taken for all starting material to be consumed.cIsolated yield after silica gel flash column chromatography.dmeta to phenol oxygen AND para to ester.eReaction was slow and incomplete after 3days.fYield based on recovered starting material.gComplex mixture of products.Table 2TFA-mediated debenzylation of O -benzyl-protected,ortho -substituted phenols aTFA tolueneOBnOH67RRSubstrate R p K aH b Time c (h)Yield d (%)Relative rate 6m CO 2NH 2À2248316n CHO À7 3.594e 6.96o CO 2H À8191246b CO 2Me À8.55min 942886d CO 2Bn À8.55min 932886p CN À10>4851(95f )—6f NO 2À1239786i Br —16—g 1.56lH—24—g1aThe reaction was carried out with 6(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt,with 3equiv of thioanisole.bApproximate p K aH of conjugate acid of R group.15cTime taken for all starting material to be consumed.dIsolated yield after silica gel flash column chromatography.eIncluding thioanisole in the deprotection of 6n led to further by-products,thus no scavenger was used and compound 7n could be obtained in only 90%purity.fYield based on recovered starting material.gComplex mixture of products.4818S.Fletcher,P.T.Gunning /Tetrahedron Letters 49(2008)4817–4819acidity of the conjugate acid (decreasing p K aH )of the ortho -elec-tron-withdrawing substituent.15There appears to be an optimal p K aH of around À8.5,that is exhibited by carboxylic esters,which lead to the fastest rate of debenzylation with TFA.In an approxi-mate bell-shaped distribution of reaction rate versus ortho -substi-tuent p K aH —that was interrupted only by ortho -cyanophenol 6p —protonatable groups with p K aH ’s <À8.5or >À8.5were less effective at accelerating the TFA-mediated debenzylation.These data concur with our chelation hypothesis:groups that are too ba-sic bind more strongly to the TFA proton making it less available for sharing with,and ultimately releasing to,the phenol ether oxygen;groups that are weakly basic do not bind the TFA proton as well,leading to reduced chelation and hence less rate enhancement.The anomalous result for ortho -cyanophenol 6p was anticipated since this compound was selected as a negative control.Phenol 6p is geometrically incapable of chelating a proton,because the lin-ear,sp -hybridized nitrile functionality directs its basic nitrogen atom (p K aH %À10)away from the phenol oxygen.As predicted,there was no rate enhancement for the TFA-mediated debenzyla-tion of 6p relative to phenol 6l .In fact,6p was only slowly deben-zylated,at a rate that was comparable with the m -nitro and p -nitro derivatives 6g and 6h ,respectively.We next wanted to investigate the selectivity for the deprotec-tion of the benzyl group over other phenol protecting groups.Accordingly,the benzyl group in salicylate derivative 9a was varied with para -methoxybenzyl (PMB;9b ),methyl (9c ),allyl (9d )and iso -propyl (i -Pr;9e ).These substrates were then debenzylated with a 1:1mixture of TFA/toluene;our findings are reported in Table 3.Any impurities this time were minor and readily separable from the products,eliminating the need for the additive thioanisole.The relative rates at which these protecting groups were removed was para -methoxybenzyl >benzyl >allyl >iso -propyl )methyl,which reflects the stability of the carbocations.These data suggest that in salicylates such as 9,the benzyl phenol protecting group (R =Bn)can be removed with TFA in the presence of the corres-ponding allyl,iso -propyl and methyl ethers.Finally,we explored the selectivity of this mild debenzylation technique over other benzyl-based protecting groups,as shown in Table 4.As the results demonstrate,it was possible to deblock the O -benzyl ether in the presence of a benzyl ester (6d )and in the presence of a benzyl carbamate (11b ),thereby increasing the orthogonality of O -benzyl phenol ethers of salicylate derivatives.Interestingly,it was even possible to cleave the benzyl group in 11c with TFA in the presence of an N -Boc-protected aniline.In summary,we have presented the mild,efficient and rapid deblocking of ortho -substituted aryl benzyl ethers with TFA.Deb-enzylation was fastest when the ortho group was a carboxylic ester,which we have attributed to a proton chelation effect.Other ortho groups that accelerated the TFA-mediated debenzylation included carboxylic acid,aldehyde and nitro.In addition,we have shown that in such ortho -functionalized phenols,benzyl could be removed in the presence of the corresponding iso -propyl,allyl and methyl ethers.Moreover,the benzyl ether could be selectively cleaved in the presence of benzyl ester,Cbz carbamate and Boc carbamate functionalities.AcknowledgementsThe authors gratefully acknowledge financial support for this work from the Canadian Foundation of Innovation and the Univer-sity of Toronto (Connaught Foundation).References and notes1.Zhang,S.;Zhang,Z.-Y.Drug Discov.Today 2007,12,373–381.2.(a)Pei,Z.;Li,X.;Liu,G.;Abad-Zapatero,C.;Lubben,T.;Zhang,T.;Ballaron,S.J.;Hutchins,C.W.;Trevillyana,J.M.;Jirouseka,M.R.Bioorg.Med.Chem.Lett.2003,13,3129–3132;(b)Xin,Z.;Liu,G.;Abad-Zapatero,C.;Pei,Z.;Szczepankiewicz,B.G.;Li,X.;Zhang,T.;Hutchins,C.W.;Hajduk,P.J.;Ballaron,S.J.;Stashko,M.A.;Lubben,T.H.;Trevillyana,J.M.;Jirouseka,M.R.Bioorg.Med.Chem.Lett.2003,13,3947–3950.3.Tautz,L.;Bruckner,S.;Sareth,S.;Alonso,A.;Bogetz,J.;Bottini,N.;Pellecchia,M.;Mustelin,T.J.Biol.Chem.2005,280,9400–9408.4.Shrestha,S.;Bhattarai,B.R.;Chang,K.J.;Leea,K.-H.;Choa,H.Bioorg.Med.Chem.Lett.2007,17,2760–2764.5.Liljebris,C.;Larsen,S.D.;Ogg,D.;Palazuk,B.J.;Bleasdale,J.E.J.Med.Chem.2002,45,1785–1798.6.Siddiquee,K.;Zhang,S.;Guida,W.C.;Blaskovich,M.A.;Greedy,B.;Lawrence,H.R.;Yip,M.L.R.;Jove,R.;Laughlin,M.M.;Lawrence,N.J.;Sebti,S.M.;Turkson,J.Proc.Natl.Acad.Sci.U.S.A.2007,104,7391–7396.7.Pandey,P.N.;Purkayastha,M.L.Synthesis 1982,876–878.8.(a)Greene,T.W.;Wuts,P.G.M.Protective Groups in Organic Synthesis ,3rd ed.;John Wiley &Sons:New York,1999;(b)Kocienski,P.J.Protecting Groups ,3rd ed.;Georg Thieme:Stuttgart,Germany,2003.9.Kiso,Y.;Isawa,H.;Kitagawa,K.;Akita,T.Chem.Pharm.Bull.1978,26,2562–2564.10.Kiso,Y.;Ukawa,K.;Nakamura,S.;Ito,K.;Akita,T.Chem.Pharm.Bull.1980,28,673–676.11.Ploypradith,P.;Cheryklin,P.;Niyomtham,N.;Bertoni,D.R.;Ruchirawat,.Lett.2007,9,2637–2640.12.Marsh,J.P.,Jr.;Goodman,.Chem.1965,30,2491–2492.13.(a)Eberle,A.N.J.Chem.Soc.,Perkin Trans.11986,361–367;(b)Bodanszky,M.;Tolle,J.C.;Deshmane,S.S.;Bodanszky,A.Int.J.Pept.Protein Res.1978,12,57–68.14.Haraldsson,G.G.;Baldwin,J.E.Tetrahedron 1997,53,215–224.15.(a)Ionization Constants of Organic Acids in Solution ;Serjeant,E.P.,Dempsey,B.,Eds.IUPAC Chemical Data Series No.23;Pergamon Press:Oxford,UK,1979;(b)see also:/labs/evans/pdf/evans_pKa_table.pdf .Table 3TFA-mediated deprotection of O-blocked phenol ether derivatives of methyl 4-acetamidosalicylate aTFAtolueneNHAcNHAcORO OMeOH OMeO 910Substrate R Time b (h)Yield c (%)9a Bn 5min 919b PMB 2min 909c Me 480d 9d Allyl 20919ei -Pr3692aThe reaction was carried out with 9(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt.bTime taken for all starting material to be consumed.cIsolated yield after silica gel flash column chromatography.dOnly starting material remained after 48h,at which point the reaction was aborted.Table 4Selectivity investigation into the TFA-mediated debenzylation of aryl benzyl ethers aTFA tolueneOBnOH2Bn2Bn1112RRSubstrate R Yield b (%)6d c H 9311a NHAc 9211b NHCbz 9311c dNHBoc54aThe reaction was carried out with 11(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt for 5min,then all solvents were evaporated.bIsolated yield after silica gel flash column chromatography.cFor compound 6d ,3equiv of thioanisole were also used.dAfter 5min,the reaction mixture was diluted with CH 2Cl 2and then immedi-ately neutralized with 1M NaOH.The organic layer was then separated and evaporated.S.Fletcher,P.T.Gunning /Tetrahedron Letters 49(2008)4817–48194819。