Isolationandstructureof26,27-cycloaplysterol(petrosterol)acyclopropane-co.

Isolation and Crystal Structure of 2—Bromoaldisin 徐效华; 陈晓; 等【期刊名称】《《结构化学》》【年(卷),期】2001(020)003【摘要】The crystal structure of the title compound (C8H7BrN2O2,Mr=243.07) was isolated from the marine sponge Phacellia fusca Schmidt collected from the South China Sea. Its crystal structure was determined by single-crystal X-ray diffraction. The crystal is orthorhombic with space group Pbca, a=12.9952(8), b=7.4479(5), c=18.598(1) ?, V=1800.1(2) ?3, Z=8, Dc=1.794g/cm3, (=0.71073 ?, ( (MoK()=4.533mm-1, F(000)=960. The structrue was refined to R=0.0349, wR(F2)=0.0925 for 1589 reflections with I > 2((I). X-ray diffraction analysis reveals that the title compound has one five-membered pyrrole ring and one seven-membered azepin ring. There are two intermolecular hydrogen bonds between two molecules.【总页数】3页(P173-175)【作者】徐效华; 陈晓; 等【作者单位】InstituteandStateKeyLaboratoryofElemento－OrganicChemistry NankaiUniversity Tianjin300071 China【正文语种】中文【中图分类】O626【相关文献】1.Isolation, Crystal Structure and Antitussive Activity of 9S,9aS-neotuberostemonine [J], WU Yi;YE Qing-Mei;LIU Jing;XU Wei;ZHU Zi-Rong;JIANG Ren-Wang2.Isolation, Crystal Structure and Na+/K+-ATPase Inhibitory Activity of 1β-Hydroxydigitoxigenin [J], XU Yun-Hui;XU Jian;JIANG Xue-Yang;CHEN Zhi-Hua;XIE Zi-Jian;JIANG Ren-Wang;FENG Feng3.Isolation and Crystal Structure of Ent-kaurane Diterpenes from Rubus corchorifolius L.f. [J], CHEN Xue-Xiang;HUANG Jian-Xi;OU Yang-Wen;LIU Xiao-Juan;ZHOU Li-Ping;CAO Yong4.Isolation, Crystal Structure,and Anti-inflammatory Activity of Sakuranetin from Populus tomentosa [J], LIU Hai-Ping;CHAO Zhi-Mao;TAN Zhi-Gao;WU Xiao-Yi;WANG Chun;SUN Wen5.Isolation and Crystal Structure of 2-Bromoaldisin [J], 徐效化; 陈晓; 廖仁安; 谢庆兰因版权原因，仅展示原文概要，查看原文内容请购买。

生防菌解淀粉芽孢杆菌抗菌蛋白的研究进展出晓铭1，2，林毅雄1，2，张珅1，2，严芬1，3，林河通1，2（1．福建农林大学食品科学学院，福州350002；2．福建农林大学农产品产后技术研究所，福州350002；3．福州大学生物科学与工程学院，福州350108）摘要：生防解淀粉芽孢杆菌具有强烈抑制真菌和细菌的能力，在果蔬采后病害防治方面具有巨大的应用潜力。

该文对解淀粉芽孢杆菌产生的抗菌蛋白、蛋白的分离纯化、抑菌机理和生防应用等方面进行了综述，并对其在果蔬采后病害防治应用前景进行了展望。

关键词：解淀粉芽孢杆菌；抗菌蛋白；采后病害；生物防治中图分类号：S476；TS255．3文献标志码：A 文章编号：1005－1295（2014）06－0049－06doi ：10．3969/j．issn．1005－1295．2014．06．013Ｒesearch Advances of Antifungal Proteins Produced by Biocontrol Bacterium ，Bacillus amyloliquefaciensCHU Xiao-ming1，2，LIN Yi-xiong 1，2，ZHANG Shen1，2，YAN Fen1，3，LIN He-tong 1，2（1．College of Food Science ，Fujian Agriculture and Forestry University ，Fuzhou 350002，China ；2．Institute of Postharvest Technology of Agricultural Products ，Fujian Agriculture and Forestry University ，Fuzhou 350002，China ；3．College of Biological Science and Engineering ，Fuzhou University ，Fuzhou 350108，China ）Abstract ：Bacillus amyloliquefaciens is a kind of biocontrol bacterium which has great ability against fungi and bacteria ，thereby it has high research values and potential for development in biological control of posthar-vest diseases of fruits and vegetables．The separation and purification of antifungal proteins produced by Bacil-lus amyloliquefaciens ，the disease resistant mechanisms and the application of biocontrol of antifungal proteins were summarized．The future potential application in biocontrol of postharvest diseases of fruits and vegetables was also discussed．Key words ：Bacillus amyloliquefaciens ；antifungal proteins ；postharvest diseases ；biological control 收稿日期：2014－06－23；修稿日期：2014－07－16基金项目：国家科技支撑计划专项（2007BAD07B06）；福建省高等学校新世纪优秀人才支持计划（闽教科〔2007〕20号）资助作者简介：出晓铭（1990－），男，硕士，研究方向为食品科学。

生物化学英语Introduction to BiochemistryBiochemistry is a fascinating interdisciplinary fieldthat combines biology and chemistry to study the chemical processes and molecules that occur within living organisms.It focuses on understanding the molecular mechanisms underlying biological processes and how they are regulated. In this document, we will provide an overview of important concepts and techniques in biochemistry.1. Structure and Function of BiomoleculesBiochemistry studies the structure and function of biomolecules, which include proteins, nucleic acids, carbohydrates, and lipids. Proteins are crucial for various cellular functions, such as enzyme catalysis, cell signaling, and structural support. Nucleic acids, including DNA and RNA, carry genetic information and are involved in protein synthesis. Carbohydrates are important energy sources, while lipids serve as components of cell membranes and energy storage molecules.2. Enzymes and MetabolismEnzymes are proteins that catalyze biochemical reactions, playing a vital role in metabolism. They lower the activation energy required for reactions to occur, thus speeding up the rate of chemical reactions within cells. Metabolism involves a series of interconnected biochemical reactions that convert nutrients into energy and building blocks for cellular processes. An understanding of enzyme kinetics and metabolic pathways is essential in biochemistry.3. Biochemical TechniquesVarious techniques are used in biochemistry to study biomolecules and their functions. These include spectroscopy, chromatography, electrophoresis, centrifugation, and molecular cloning. Spectroscopy allows the analysis of biomolecule structures by using light absorption, emission, or scattering. Chromatography separates mixtures into their individual components. Electrophoresis separates charged molecules based on their size and charge. Centrifugation separates particles based on their size and density. Molecular cloning allows for the replication and manipulation of DNA.4. Gene Expression and RegulationBiochemistry also encompasses the study of gene expression and regulation. Gene expression refers to the process by which information from a gene is used to produce a functional protein or RNA molecule. Regulation of gene expression ensures that the right genes are turned on or off at the appropriate times and in specific cell types. Understanding gene expression and regulation is crucial in understanding development, cell differentiation, and disease.5. Applications of BiochemistryBiochemistry has numerous applications in various fields, including medicine, agriculture, and biotechnology. In medicine, biochemistry is essential for understanding diseases at the molecular level and developing new drugs and therapies. In agriculture, biochemistry is used to improve crop yields and develop genetically modified organisms. Biotechnology relies heavily on biochemistry for geneticengineering, production of recombinant proteins, and designing new biofuels.ConclusionBiochemistry is a vast and dynamic field that plays a critical role in advancing our understanding of life processes and their applications. It provides a foundation for various other branches of biology and chemistry, contributing to fields such as molecular biology, genetics, and pharmacology. By studying the structure and function of biomolecules, enzymes, and metabolic pathways, biochemists continue to unravel the complexities of life.。

周桂成，肖珊，王波，等. 酪蛋白肽锌螯合物的制备及体外消化分析[J]. 食品工业科技，2023，44（23）：270−279. doi:10.13386/j.issn1002-0306.2023020002ZHOU Guicheng, XIAO Shan, WANG Bo, et al. Preparation and in Vitro Digestive Analysis of Casein-Derived Peptide-Zinc Chelates[J]. Science and Technology of Food Industry, 2023, 44(23): 270−279. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023020002· 分析检测 ·酪蛋白肽锌螯合物的制备及体外消化分析周桂成1,2，肖　珊2，王　波2, *，王际辉2,*（1.大连工业大学生物工程学院，辽宁大连 116034；2.东莞理工学院生命健康技术学院，广东东莞 523808）摘　要：为开发安全高效且易吸收的补锌剂，利用碱性蛋白酶酶解和乳酸菌发酵相结合的方法制备生物活性肽，并以此多肽制备了酪蛋白肽锌螯合物。

采用光谱法对螯合物结构进行表征，利用体外消化模型和Caco-2细胞实验对其胃肠消化特性及生物安全性进行评价。

结果表明，制备酪蛋白肽的最优条件为酶解pH 为9、碱性蛋白酶添加量为0.3%（w/v ），乳酸菌发酵时间为12 h ，此时反应体系中多肽含量为142.39±0.95 mg/g ，对锌的螯合率为31.41%±0.97%。

与锌螯合后，酪蛋白肽表面的致密结构遭到破坏，形成疏松的状态；光谱学分析表明，Zn 2+能与酪蛋白肽上的活性基团进行结合，螯合位点为羧基氧、羟基氧和氨基。

体外模拟消化结果显示，酪蛋白肽锌螯合物在消化过程中锌溶解性优于硫酸锌；在胃肠消化后酪蛋白肽锌螯合物DPPH 和ABTS +自由基的清除能力分别提升了26.19%±3.30%和71.96%±7.06%，而铁还原力下降了36.26%±2.80%；同时，在消化过程中肽锌螯合物的β-转角与无规则卷曲含量减少，β-折叠结构增加，Zn 2+起到了维持多肽结构的作用。

收稿日期:2003-10-17基金项目:国家自然科学基金资助项目(50170034)(30170026)作者简介:刘 缨(1976-),女,助理工程师,主要从事自养微生物的分子生物学研究.文章编号:1671-9352(2004)02-0116-04一株螺旋状铁氧化细菌的分离及特性研究刘 缨,刘相梅,郑力真,林建群,颜望明(山东大学 微生物技术国家重点实验室,山东 济南 250100)摘要:利用双层平板培养技术,从云南腾冲地区高温温泉边酸性水中分离出1株螺旋状铁氧化细菌ML -04,对该菌的理化特性研究结果显示,该菌专性化能自养,可利用亚铁和黄铁矿为能源,不能氧化硫磺、四硫酸盐、硫代硫酸盐,最适生长温度40e ,最适生长p H2.5.对砷黄铁矿的浸矿实验表明,ML -04菌株可以有效浸出矿粉中的铁和硫元素.关键词:螺旋状;铁氧化细菌;分离;理化特性中图分类号:Q93 文献标识码:AIsolation and characterization of a vibrioid -shaped iron -oxidizing bacteriumLIU Ying,LIU Xiang -mei,Z HENG L-i zheng,LIN Jian -qun &YAN Wang -ming(State Key Laboratory of Microbial Technol ogy,Shandong Univ.,Ji nan 250100,Shandong,China)Abstract :A vibrioid -shaped i ron -oxidizing bacteriu m,named strain ML -04,was isolated from Tenchong area,Yunnan province in China with the double layer culture technique.And the characterization resul ts showed that ML -04s train is obli gately autotrophic and could use ferrous iron and p yrite as sole energy sources,but not element sulfur,thiosulfate and tetrathionate.The opti mal tem -perature of ML -04strai n is 40e and the opti mal p H is 2.5for growth.The result of leaching test showed that iron and sulfur ele -ment could be effectively extracted from arsenopyrite by bio -oxidation process of the ML -04strain.Key words :Vibrioid -shaped;Iron -oxidizing bacterium;Isolati on;Physi ological characters生物浸矿技术具有悠久的历史,古代人们就用微生物浸出铜.和化学方法相比,生物浸矿具有多方面的优点,不仅耗能低,对环境污染小,还可以处理常规化学方法难处理的低品位矿石,因而在环境问题日益严重,金属富矿匮乏的今天,越来越受到人们的关注[1].生物浸矿技术在一些国家已成功应用于工业生产,如南非、巴西、澳大利亚、美国、加纳、秘鲁、乌兹别克斯坦、希腊等国都实现了生物浸矿的工业化应用.我国在这方面的工作也开始起步,2000年,山东莱州天承生物金业股份有限公司引进澳大利亚生物提金技术处理含砷难冶金精矿,日处理矿石能力可达120吨[2].浸矿微生物主要是一些在酸性环境中生长的铁或硫氧化细菌.多为化能自养,可利用低价态铁或还原态无机硫化物作为电子供体,具有嗜酸性,生长pH 在1.5~2.0左右[3].目前国外已广泛开展对浸矿细菌的研究,以更好的应用于生产实践.但研究过程中存在很多技术难点,尤其是浸矿细菌绝大多数都是专性化能自养,有机质的存在对其生长有抑制作用,因而在琼脂糖或琼脂固体培养基上难以生长,也就难以对其进行分离纯化.目前分离纯化这类细菌多采用双层平板培养技术,该技术是在底层培养基中加入1种嗜酸性异养细菌,通过底层异养细菌的生长,消耗了琼脂第39卷 第2期Vol.39 No.2山 东 大 学 学 报 (理 学 版)JOURNAL OF SHANDONG UNI VERSITY2004年4月 Apr.2004糖固体平板中的微量有机成分,如寡糖类物质,从而有利于上层平板中化能自养细菌的生长[4,5].本研究利用双层平板培养基技术,从采集自云南腾冲地区高温温泉边的酸性水样中分离纯化出1株螺旋状铁氧化细菌ML-04,对该菌的理化特性和浸矿能力进行了研究.1材料1.1样品采自云南腾冲地区高温温泉边酸性水样.1.2培养基ML-04菌株的分离用FeTSB双层平板培养基[5]、生长曲线的测定用9K矿粉培养基[6],即9K培养基中加入砷黄铁矿(该矿粉含S20.45%,Fe 24.9%,As5.16%),用1mol P L的硫酸调pH至2.5.ML-04菌株能源利用特性的测定用基础培养基[7].该培养基用1mol P L的硫酸调pH至2.5.2方法2.1ML-04菌株的分离将采集的样本在9K培养基中,37e富集培养10d左右,待培养基颜色变成红棕色,采用梯度稀释法,涂布Fe TSB双层平板培养基,37e培养7d左右,长出单菌落,挑取单菌落在Fe TSB双层平板培养基上连续分离纯化,镜检观察,直至菌体形态一致.记作ML-04.收集ML-04菌体,经革兰氏染色后在光学显微镜下观察并摄影.收集ML-04菌体,涂布在小块盖玻片上,自然晾干后,用导电胶贴于圆形贴片上,喷金后在扫描电镜下观察及摄影.2.2ML-04菌株最适生长温度的测定以相同接种量接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,分别置于25e、35e、40e、45e、55e五个温度梯度,摇床培养6d,用血球计数板计菌数,以温度为横坐标,以菌数的对数值为纵坐标,绘制ML-04的生长温度)))菌数曲线图.2.3初始pH对ML-04菌株生长的影响以相同接种量接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,培养基pH值分别用奥力龙828型pH计标定至0.5,1.5,2.5,3.5,4.5,40e摇床培养6d,用血球计数板计菌数,以初始pH为横坐标,以菌数的对数值为纵坐标,绘制ML-04的pH)))菌数曲线图.2.4ML-04菌株生长曲线的绘制接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,pH2.5,40e条件下摇床培养,转速120r P min,每隔2d用血球计数板计菌数,以培养天数为横坐标,以菌数的对数值为纵坐标,绘制ML-04的生长曲线图.2.5ML-04菌株能源利用特性的研究向基础培养基中分别加入蛋白胨(0.1%)、酵母粉(0.1%)、葡萄糖(0.1%)、硫磺(5%)、硫代硫酸钠(1%)、四硫酸钾(0.3%)、硫酸亚铁(0.1mol P L)和黄铁矿(5%).其中硫代硫酸钠、四硫酸钾、硫酸亚铁过滤除菌后加入培养基,硫磺隔水蒸煮1h灭菌,再加入培养基.以ML-04菌悬液接种,40e摇床培养6d,连续3代移种,在显微镜下涂片观察菌的生长情况,生长者为阳性.2.6ML-04氧化砷黄铁矿的研究接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,每隔2d测1次培养基中可溶性总铁的量及硫酸根离子的量.总铁的测定用邻二氮菲分光光度法,硫酸根离子的测定用比浊法[8].以溶液中可溶性总铁的量与培养基中矿粉总铁含量的比例为纵坐标,以培养天数为横坐标,绘制铁的氧化率曲线.以溶液中硫酸根离子含量与培养基中矿粉含硫总量的比例为纵坐标,以培养天数为横坐标,绘制硫的氧化率曲线.3结果3.1菌株的分离及形态学观察菌株ML-04的菌落形态为琥珀色同心圆状,直径1~3mm,表面湿润,凸起,光滑(见图1).菌体形态为螺旋状,大小(0.25~0.3)@(1~3L m),革兰氏染色阴性(见图2和图3).3.2ML-04菌株最适生长温度的测定由图4可以看出,ML-04最适生长温度在40e 左右.当培养温度达到55e时,ML-04的菌数迅速下降.3.3初始pH对ML-04菌株生长的影响从图5可见,ML-04菌株生长的最适初始pH为2.5左右.在此pH条件下生长最好.第2期刘缨,等:一株螺旋状铁氧化细菌的分离及特性研究117图1 ML -04菌株的菌落形态照片Fig.1 The Colony of ML -04图2 ML -04菌株的光学显微镜照片(@1000)Fig.2 M icrograph of ML -04图3 ML -04菌株的扫描电镜照片(@15000)Fig.3 Scanning electron micrograp h of ML -04图4 培养温度对ML -04生长的影响Fig.4 Effect of temperature on the growth of ML -04strain图5 初始pH 对ML -04生长的影响Fig.5 Effect of culture media p H on the growth of ML -04strain3.4 ML -04菌株的生长曲线从图6可见,ML -04经过4d 左右的生长延滞期,4~6d 为对数生长期,从第8天以后,菌数增长非常缓慢.图6 ML -04菌株的生长曲线Fig.6 The growth curve of ML -04strain3.5 ML -04能源利用特性的研究由表1可见,ML -04菌株可以利用硫酸亚铁和黄铁矿为能源生长,不能利用硫磺、硫代硫酸盐和四硫酸盐,也不能利用葡萄糖、蛋白胨等有机物.表1 ML -04能源利用特性的研究T ab.1 Energy sources utilization characters of ML -04strai n能源生长情况蛋白胨-酵母粉-葡萄糖-硫磺-硫代硫酸钠-四硫酸钾-硫酸亚铁+黄铁矿+3.6 ML -04菌株对砷黄铁矿中铁和硫的氧化浸出结果ML -04菌株对铁的氧化浸出率见图7.ML -04菌株可以有效浸出砷黄铁矿中的铁,随着培养天数的增加,溶液中总铁的含量不断提高,第18天时,铁的浸出率在90%以上.图7 ML -04菌株对砷黄铁矿中铁的氧化浸出曲线Fig.7 The curve of iron concentration in the extraction ofarsenopyrite by ML -04strain118山 东 大 学 学 报 (理 学 版)第39卷ML -04菌株对砷黄铁矿中硫的氧化浸出率见图8.随着培养天数的增加,溶液中硫酸根的含量不断提高,第18天时,硫的浸出率达80%以上.图8 ML -04菌株对砷黄铁矿中硫的氧化浸出曲线Fig.8 The curve of sul fur concentration in the extraction ofarsenopyrite by ML -04s train4 讨论长期以来,人们一直认为氧化亚铁硫杆菌(Thiabacillus ferrooxidans )在浸矿过程中起主要作用,近些年的研究却发现,钩端螺旋菌属(Leptos pirillum )在浸矿过程中往往起关键作用.和T .ferroo xidans 相比,Leptospirillum 属菌株如氧化亚铁钩端螺旋菌(L .ferroo xidans )的氧化还原电势更高,对铁离子的耐受能力更强,生长不受高浓度三价铁离子的抑制.因而,在工业生产中,尤其是在连续反应浸矿系统中,Leptos pirillum 属菌株占微生物种群的主要组分,在浸矿过程中起主导作用[9,10].因此,有关钩端螺旋菌属资源的发掘及其浸矿特性的研究,对微生物浸矿技术的发展和应用具有十分重要的意义.本实验的研究结果表明,ML -04菌株是螺旋状铁氧化细菌.它的最适生长温度40e ,最适生长pH2.5,和Leptos pirillum 属的L .ferrooxidans 一样,既能氧化硫酸亚铁,又能氧化硫化矿物,不能氧化硫磺、硫代硫酸盐及四硫酸盐.不能利用有机物为能源进行生长,属于化能自养型微生物[3,11].ML -04菌株与T .ferroo xidans 和L .ferroo xidans 的比较见表2.铁氧化细菌中的氧化亚铁钩端螺旋菌(L .fer -roo xidans )虽然不能直接利用无机硫化物作为能源,但它们在矿物的生物氧化过程中,通过浸出矿物中的铁元素,产生的硫酸铁中间代谢物是一种有效的金属矿物氧化剂,可作用于矿物中的无机硫,将硫元素也浸提出来[12].对ML -04的浸矿实验研究表明,ML -04菌株可有效浸出砷黄铁矿中的铁和硫.因而,该菌株在实际生产中具有潜在的应用价值,有关该菌株更为广泛的浸矿特性研究,本室正在进一步进行中.表2 ML -04菌株与T .f e rrooxidans 和L .f e rrooxidans的比较T ab.2 Characteristics of Strain ML -04,T .ferrooxidans andL .ferrooxidans生理生化特性ML -04L .ferrooxidan T .ferrooxidan s 革兰氏染色G -G -G -菌体形态螺旋状螺旋状杆状菌体大小(L m)0.25~0.3@1~30.2~0.4@1~30.5~1@1~2最适生长温度(e )4037~4030最适生长pH 2.5 1.5~1.8 2.5~3.0生长类型化能自养化能自养化能自养能量来源Fe 2+Fe 2+Fe 2+,S 0参考文献:[1]童雄.微生物浸矿的理论与实践[M].北京:冶金工业出版社,1997.[2]杨显万,郭玉霞.生物湿法冶金的回顾与展望[J].云南冶金,2002,31(3):85~88.[3]Rawlings D E.Heavy metal mining using microbes[J].AnnuRev Microbi ol,2002,56:65~91.[4]Johnson D B,Macvicar J H M,Rolfe S.A new solid mediumfor the i solatation and enumeration of Thiabacillus ferrooxidan s and acidophilic heterotrophic bacteria [J].J Microbial M eth -ods,1987,7:9~18.[5]Johnson D B,M cGinness S.A highly efficient and universalsolid medium for growing mesophilic and moderately thermo -philic,iron -oxidizi ng ,acidophilic bacteria [J ].J M icrobial Methods,1991,13:113~122.[6]Silverman M P,Lundgren D G.Studies on the chemoautotro -phic iron rium Ferr obacterium f e rrooxidans I:An improved me -dium and harvesting procedure for securing high cell yields[J].J Bacterial,1959,77:642~647.[7]东秀珠,蔡妙英等.常见细菌系统鉴定手册[M].北京:科学出版社,2001.[8]南京大学5无机及分析化学实验6编写组.无机及分析化学实验(第三版)[M ].北京:高等教育出版社,1998.[9]Rawlings D E,Tributsch H,Hansford G S.Reasons why-Le ptospirillum .-like species rather than Thiobacillus f err ooxi -dans are the dominant iron -oxidizing bacteria in many commer -cial processes for the biooxidati on of pyri te and related ores[J].Microbi ology,1999,145:5~13.(下转第124页)第2期刘 缨,等:一株螺旋状铁氧化细菌的分离及特性研究119点:1、ZAP Express载体具有包括EcoRÑ、NotÑ在内的12个单一酶切位点,可插入0-12kb的DNA片段;2、在克隆位点两侧,含有T3、T7、Lac、C MV等双向启动子满足在原核和真核中表达的条件;3、具有T3、T7等多条测序引物;4、含有Ne o r-Kan r抗性标记便于重组子的筛选;5、构建于该载体上的克隆可用DNA探针或抗体探针筛选;7、由于ZAP E xpress中引入了f1噬菌体的复制其始和终止信号,在得到阳性克隆噬菌斑后,利用辅助噬菌体E xAssist进行超感染,使插入片段连同pB K-C MV从噬菌体DNA上剪切下来,形成噬菌粒,省掉了插入片段从噬菌体DNA到质粒载体上的酶切、连接和转化这一过程,极大方便了在体外对插入DNA片段的亚克隆操作.关于ZAP E xpress更为详尽的讨论见文献[5].利用分离纯化的火菇素的免疫血清对金针菇表达型cDNA文库进行免疫筛选,再对阳性克隆用PC R、限制性内切酶进行酶切、大肠杆菌初步诱导分析,获得了目的基因相关的cDNA片段,进一步的序列分析、鉴定和克隆火菇素基因的工作正在进行之中.参考文献:[1]Komatsu N,T erakawa H,Nakanishi K.Flammulin,a basicprotein of Flammulina velutipes wi th ant-i tumor activities[J].Antibiotics,Ser A,1963,16(3):139~143.[2]Watanabe Y,Nakanishi K,Komaisu N.Flammulin,an ant-itumor substance[J].Bull Chem Soc,Japan,1964,37(5): 747~750.[3]周凯松,彭俊峰,张长铠,等.火菇素提取新工艺及其生物活性研究[J].中国生物化学与分子生物学报, 2003,(2),In press.[4]张龙翔.生化实验方法和技术[M].北京:高等教育出版社,1997.[5]Joseph Sambrook,David W.Russel.分子克隆(第三版)[M].黄培堂,等译.北京:科学出版社,2002.[6]Laemmli UK.Cleavage of structural proteins during the assem-bly of the head of bacteriophage T4[J].Nature,1970,227: 680~685.(编辑:于善清)(上接第119页)[10]Sand W,Rohde K,Sobotke B,et al.Evaluation of Leptospi-rillu m f err ooxidans for leaching[J].Appl Environ Microbiol, 1992,58:85~92.[11]Arthur P,Harrison J.Genomic and physiological diversi tyamongst s trains of Thiobacillus ferrooxidand,and genomic comparison wi th Thiobacillus thioox idans[J].Arch Microbial,1982,131:68~79.[12]Battaglia-Brunet F,d,Hugues P,Cabral T,et al.T he mutu-al effect of mixed Thiobacilli and Leptospirilli populations on pyrite bioleachina[J].Minerals Engineering,1998,11(2): 195~205.(编辑:于善清)124山东大学学报(理学版)第39卷。

生物技术通报BIOTECHNOLOGY BULLETIN2009年第3期·综述与专论·收稿日期：2008-09-18作者简介：郭慧（1983-），女，在读硕士研究生，研究方向：动物分子生物学；E -mail ：hupoahui@ 通讯作者：张映（1954-），女，教授，研究方向：动物分子生物学糖蛋白广泛存在于生物体内，具有很多重要功能。

糖蛋白作为细胞信息功能的承担者，既是激素、凝集素、酶、毒素、病毒和细菌等的识别点，又是细胞表面抗原、糖分化抗原和癌发育抗原。

糖蛋白上的糖链好像细胞表面的天线，是细胞相互识别、粘着、信号接收、免疫应答、接触抑制、细胞分化、增殖以及受体功能等的分子基础[1]。

1糖蛋白的结构糖蛋白是由长度较短，带分支的寡糖与多肽链共价连接而形成。

糖蛋白的糖链可以是直链和分支链，糖基数一般1～15个左右，但也有含20个糖基的巨寡糖。

不同糖蛋白分子中，其糖链数目不等，分布亦不均。

如膜糖蛋白的糖链全部分布在暴露于膜外侧面的肽链上，理论上讲，糖链有无数种结构形式[2]。

然而，生物体内有某种限制因素，使实际存在的糖链类型大减，分为两类糖链，即N -连接的糖链和O -连接的糖链。

1.1N -连接的糖链N -糖链根据五糖核心结构（N -连接的糖蛋白链均含此结构）连接其它糖的情况，可分3类：高甘露糖型：寡糖链只含有甘露糖和N -乙酰氨基葡萄糖，而且只有甘露糖连接在五糖核心区上，如卵蛋白。

复杂型：寡糖链除含有甘露糖和N -乙酰氨基葡萄糖外，还有半乳糖、岩藻糖和唾液酸等。

杂合型（混合型）：既有高甘露糖链，又有N -乙酰氨基半乳糖链连在五糖核心结构上。

1.2O -连接的糖链O -连接的糖链存在多种形式，其结构共同点是由一个或少数几种单糖与某些含羟氨基酸连接，不存在共有的核心结构。

但在O -乙酰半乳糖胺（O -Gal NAC ）连接的糖链中已发现有4种核心结构，研究最多的是粘蛋白血浆蛋白和膜蛋白[3]。

山东农业科学2014，46（5）：7 11，30Shandong Agricultural Sciences收稿日期：2014－02－20基金项目：现代农业产业技术体系建设专项（CAＲS －25）作者简介：赵军林（1989－），男，硕士研究生，研究方向为蔬菜种质资源创新与生物技术。

E －mail ：zhaojlin2012@163．com *通讯作者，E －mail ：xfwang@sdau．edu．cn橙色果肉甜瓜β－胡萝卜素积累的分子机理赵军林，于喜艳，王秀峰*（山东农业大学园艺科学与工程学院/作物生物学国家重点实验室/农业部黄淮地区园艺作物生物学与种质创制重点实验室，山东泰安271018）摘要：采用HPLC 分别测定了橙色果肉甜瓜Homoka 和对照白色果肉甜瓜M01－3六个发育时期的β－胡萝卜素及叶黄素含量，并对相关基因作了生物信息学及表达分析。

结果表明：随果实发育，橙色甜瓜β－胡萝卜素含量显著升高，在接近成熟时达到积累高峰，成熟时又有所降低；两种甜瓜果实中β－胡萝卜素合成相关基因PSY2、PDS 、ZDS 、LCY －b 的表达量均升高，但橙色甜瓜中PDS 和LCY －b 表达量高于白色甜瓜；β－胡萝卜素的裂解酶基因CCD1在橙色甜瓜中表达下调，而在白色甜瓜中上调。

与白色果肉甜瓜M01－3相比，PDS 、LCY －b 的高表达和CCD1表达的下调可能决定了橙色甜瓜果实中β－胡萝卜素的高积累量。

关键词：甜瓜；果实；β－胡萝卜素；积累中图分类号：Q786文献标识号：A文章编号：1001－4942（2014）05－0007－06Molecular Mechanism of β－Carotene Accumulationin Orange －Fleshed MuskmelonZhao Junlin ，Yu Xiyan ，Wang Xiufeng *（College of Horticulture Science and Engineering ，Shandong Agricultural University /State Key Laboratory of Crop Biology /Key Laboratory of Biology and Genetic Improvement of HorticulturalCrops ，Huanghuai Ｒegion ，Ministry of Agriculture ，P．Ｒ．China ，Taian 271018，China ）AbstractThe HPLC method was used to determine the contents of β－carotene and lutein at six devel-opmental stages in orange －fleshed muskmelon Homoka with white －fleshed muskmelon M01－3as control．And the bioinformatics and expression analyses of related genes were made．The results showed that the con-tent of β－carotene in orange －fleshed muskmelon increased significantly with the development of fruits andreached the accumulation peak when close to maturity ，but declined somewhat in mature fruit．The expression level of genes related to biosynthesis of β－carotene ，PSY2，PDS ，ZDS ，LCY －b ，were all up －regulated in two muskmelon fruits．But the expression quantity of PDS and LCY －b in orange －fleshed muskmelon was higher than that of white －flashed muskmelon．The expression level of lyase gene of β－carotene ，CCD1，de-scended in orange －fleshed muskmelon ，while ascended in white muskmelon．Compared with white －flashed muskmelon ，the high expression level of PDS and LCY －b and down －regulated expression of CCD1possibly determined the high accumulation of β－carotene in orange －fleshed muskmelon fruits．Key words Muskmelon ；Fruit ；β－carotene ；Accumulation 甜瓜（Cucumis melo L．）是世界重要的园艺作物，其果实营养丰富、芳香味浓郁、口感良好，深受消费者欢迎。

农业环境科学学报２００8,２7(6):2423-2429ＪｏｕｒｎａｌｏｆＡｇｒｏ-ＥｎｖｉｒｏｎｍｅｎｔＳｃｉｅｎｃｅ摘要：采用20种不同培养基分离水葫芦内生细菌，利用细菌脂肪酸鉴定技术对水葫芦的内生细菌进行鉴定，研究了水葫芦内生细菌的种类及群落结构。

结果表明，共得到32个属56株内生细菌，种类最多的是微杆菌属（Microbacterium ），共有9种细菌。

其次为假单胞菌属（Pseudomonas ），共有7种细菌。

芽孢杆菌属（Bacillus ）有5种细菌。

其余23个属均有1种。

利用优势度指数分析各培养基分离到内生细菌的多样性，10号葡萄糖、酵母、淀粉琼脂培养基优势度指数为0.1607，分离得到内生细菌种类最多为9种；其次是4号根瘤菌培养基和11号NA 培养基的优势度指数为0.1250，分离得到的内生菌种类均为7种；16号黄豆芽汁培养基（pH7.2~7.4）和20号醋酸菌培养基，优势度指数为0.0893，分离获得内生细菌均为5种。

关键词：水葫芦；内生细菌中图分类号：X172文献标识码：A文章编号：1672-2043(2008)06-2423-07收稿日期：2007-12-26基金项目：福建省发改委重大项目（闽发高技[2005]1061号）；福建省财政专项-福建省农业科学院科技创新团队建设基金（STIF-Y03）作者简介：蓝江林（1972—），副研究员，研究方向为生物技术与生物防治。

E-mail ：lanjianglin2002@通讯作者：刘波E-mail ：fzliubo@水葫芦内生细菌的分离与鉴定蓝江林，朱育菁，苏明星，葛慈斌，刘芸，刘波（福建省农业科学院农业生物资源研究所，福建福州350003）Isolation and Identification of the Endophyte Bacteria from Eichhornia crassipe （Mart.）Solms ．LAN Jiang-lin,ZHU Yu-jing,SU Ming-xing,GE Ci-bin,LIU Yun,LIU Bo（Agricultural Bioresource Research Institute ，Fujian Academy of Agricultural Sciences,Fuzhou 350003,China ）Abstract ：Endophyte bacteria were isolated from the plant tissues of Eichhornia crassipe （Mart.）Solms by using 20types of cultural media.The collected 56bacterial isolates were identified with bacterial fatty-acid identification system to study the population structure of endo －phyte bacteria inside E.crassipe .The endophyte bacteria were classified into 32genera,while most belonged to Microbacterium with 9iso －lates,Pseudomonas with 7isolates and Bacillus with 5isolatesa as well as the other 23genus had only 1isolate respectively.Study diversity of endiophyte bacterica on culture medium using dominance index,result showed dominance index of glucose,yeast,starch and agar culture medium is 0.1607,endophyte bacteria is 9species.Dominance index of Rhizobiumv culture medium and NA culture medium was 0.1250,had 7isolate respectively.Next was soybean sprouts juice culture medium （pH 7.2~7.4）and Acetobacter Aceti culture medium,dominance index was 0.0893,endophyte bacteria was 5species.Keywords ：Eichhornia crassipe （Mart.）Solms;endophyte bacteria植物内生细菌（Endophyte bacteria ）是指能在健康植物组织内栖居，对植物不造成实质性危害而与植物建立了和谐联合（compatible association ）关系的微生物[1]。

近年来研究证实：在脑肿瘤组织及其细胞系中存在脑肿瘤干细胞（brain tumor stem cells，BTSC），其为引发肿瘤并维持脑肿瘤生长和复发的细胞来源，已成为神经外科研究的热点问题之一[1-5]。

我们从人脑胶质母细胞瘤组织中成功分离出BTSC，并进行体外培养、鉴定和生物学特性的初步研究，现报告如下。

1材料与方法1.1实验材料1.1.1试剂：DMEM-F12、B27购自Gibco公司；表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）购自PeproTech公司；胎牛血清（FBS）、胰酶、Cy3标记的绵羊抗兔IgG均购自Sigma公司；兔抗人CD133抗体购自Abcam公司；兔抗人神经元特异性烯醇化酶（NSE）抗体、兔抗人胶质纤维酸性蛋白（GFAP）抗体、异硫氰酸荧光素（FITC）标记的羊抗兔IgG均购自武汉博士德公司。

1.1.2仪器：德国Heraeus and LISHEN公司BB16型CO2恒温恒湿培养箱及HF-safe-1200型超净工作台，日本Olympus公司CKX41型倒置相差显微镜及BX51型荧光显微镜和成像系统。

1.1.3材料来源：6例胶质母细胞瘤标本组织取自2007年12月~2008年4月在我院手术的病人，并经病理诊断为胶质母细胞瘤（WHOⅣ级）。

胶质母细胞瘤肿瘤干细胞的分离培养与生物学特性研究牛朝诗，倪永丰，陈建民（安徽医科大学附属省立医院神经外科安徽省立体定向神经外科研究所，安徽合肥230001）摘要：目的从人脑胶质母细胞瘤中分离培养脑肿瘤干细胞（brain tumor stem cells，BTSC），并研究其生物学特性。

方法利用无血清培养基和悬浮培养方法，从6例人胶质母细胞瘤标本中分离培养BTSC，并通过单克隆形成实验观察脑肿瘤干细胞球（brain tumor sphere，BTS）的形成过程。

将BTSC接种于含血清培养基，观察其在体外的分化特点。

将BTSC子代分化细胞更换培养条件，观察其在无血清培养基中的逆向分化现象。

同倍杂交种高山松与亲本种云南松的地理隔离研究*刘永良1,毛建丰2,王晓茹2,李　悦1**(1北京林业大学林木育种国家工程实验室,教育部林木花卉遗传育种重点实验室,北京　100083;2中国科学院植物研究所系统与进化植物学国家重点实验室,北京　100093)摘要:为了探讨横断山区同倍杂交种高山松与其亲本种云南松的地理隔离机制,采用实际的路线踏查方法,调查了高山松和云南松群体的地理分布情况和开花物候特征,并基于7个气象因子进行了聚类分析㊂调查研究表明高山松和云南松群体分布在不同的地貌类型中,以云南中甸为临界区,沿着金沙江河谷向北分布高山松,向南分布云南松㊂云南松的开花物候日期明显早于高山松,聚类分析表明两个物种分别聚为一支㊂在地质历史过程中,高大山系的形成以及海拔的升高将高山松和云南松从地理上隔离开来,两物种的适生区的气象因子的差异造成花期不遇,从而阻碍了两个物种间的基因交流,进而隔离开来㊂关键词:高山松;云南松;地理分布;物候;生态分化;隔离中图分类号:Q 948.2 文献标识码:A 文章编号:2095-0845(2011)03-269-06Geographic Isolation between the Homoploid HybridPinus densata and Its Parental Pinus yunnanensisLIU Yong⁃Liang 1,MAO Jian⁃Feng 2,WANG Xiao⁃Ru 2,LI Yue 1**(1Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants ,National Engineering Laboratory of Forestry Breeding ,Beijing Forestry University ,Beijing 100083,China;2State Key Laboratory of Systematic and Evolutionary Botany ,Institute of Botany ,Chinese Academy of Sciences ,Beijing 100093,China)Abstract :In order to elucidate the geographic isolation mechanisms between the homoploid hybrid Pinus densata and its parental Pinus yunnanensis in the Hengduan Mountains,we investigated the geographical distribution and flowering phenology of the populations of P.densata and P.yunnanensis by the actual route survey.The cluster anal⁃ysis was based on seven meteorological factors.The resutls showed that P.densata and P.yunnanensis were distribu⁃ted in different landscapes,and Zhongdian which located in Yunnan Province was the distribution critical region be⁃tween the two species.P.densata was distributed northwardly along the Jinshajiang valley,and P.yunnanensis southwardly.The flowering phenology of P.yunnanensis was earlier than that of P.densata .The UPGMA clustering of the populations produced two clearly defined groups,almost all the P.densata populations forming one cluster and P.yunnanensis another.Because of the apophysis of the high mountains and altitude arising in geological history,P.densata and P.yunnanensis were separated geographically.The difference of meteorological factors in suitable regions for each species made their flowering phenology not meet,which blocked the gene flow between them,and formed the isolation.Key words :Pinus densata ;Pinus yunnanensis ;Geographic distribution;Flowering phenology;Ecological diver⁃gence;Isolation植物分类与资源学报　2011,33(3):269~274Plant Diversity and Resources DOI :10.3724/SP.J.1143.2011.10227***基金项目:国家自然科学基金(30830010,31070591)通讯作者:Author for correspondence;E⁃mail:liyue@收稿日期:2010-12-10,2011-01-06接受发表作者简介:刘永良(1985-)男,在读硕士研究生,主要研究森林遗传学方面工作㊂E⁃mail:ysualiang@　高山松(Pinus densata)是现在唯一被分子生物学证明为同倍体杂交起源的木本裸子植物种,广泛分布于我国四川西部㊁西藏东部和云南西北部高山地区,是横断山区森林的建成种之一㊂高山松的形态和解剖性状介于云南松(P. yunnanensis)和油松(P.tabulaeformis)之间(Fu 等,1999),因此推断高山松可能为云南松和油松的杂种(吴中伦,1956;管中天,1981)㊂高山松杂种起源的首次遗传学证据来自于等位酶和叶绿体DNA研究(Wang和Szmidt,1994;Wang等, 2001;Liu等,2003;Song等,2003)㊂地理隔离是物种间基因交流的天然障碍,也是影响群体间遗传分化的重要因素(Schwanitz, 1969)㊂地理隔离是指由于环境变化,某物种较为连续的分布区被分成几个分布区后,各分布区种群也就随之被分隔开来㊂地壳运动㊁气候变迁等环境发生改变,产生了新的阻碍因素,导致分布区发生隔离㊂在地理隔离初期,各亚种群仍旧保持祖先种群的遗传组成,它们与当地的生态环境发生相互作用,经过长期的适应性进化最终产生分化(Widmer等,2008;Ma等,2010)㊂以往的研究多集中于证明高山松是云南松和油松的同倍性杂交物种,来自分子生物学和形态学的证据无外乎都在说明这一问题㊂本文旨在调查高山松和云南松的地理分布格局和开花物候特征,并结合气象因子来探讨两物种间可能的地理隔离机制㊂1　材料与方法1.1　研究区概况横断山区具有特殊的地理位置和复杂多样的生境,长期以来受到生态学家们的关注(Frenzel等,2003)㊂横断山区地理范围包括四川西部㊁云南西北部和西藏东部㊂该地区是我国第一大台阶 青藏高原跨入第二大台阶 云贵高原的过渡区域㊂地形特点是受大地构造的控制,形成大体平行㊁南北向延伸的高山与峡谷㊂横断山区是全国河流密度最大的地区,怒江㊁澜沧江和金沙江南北流向将高原深度切割㊂高原地形经南北流向的3条大江及其纵横交错的支流将横断山区众多的山峰彼此隔离,形成了典型的 岛屿式”生境㊂横断山区在全国气候分区上跨越热带㊁亚热带和高原寒带3个气候带,但由于本区复杂的山地地形,局地气候变化多端(Chang,1981)㊂1.2　物种分布及开花物候信息收集在野外进行了踏查工作,依据高山松和云南松的形态学特征(表1)来判定物种㊂通过中国数字植物标本馆(CVH;/)提取了云南松和高山松的标本信息,同时查阅了‘西南高山林区森林综合考察报告“(王宝田和潘志刚,1963),‘四川植被“(四川植被协作组,1980),‘云南植被“(云南植被编写组,1987),‘西藏植被“(陈伟烈,1988),‘横断山区自然地理“(张荣祖,1997)和‘青藏高原维管植物及其生态地理分布“(吴玉虎,2008)等地方植被研究资料来确定云南松和高山松地理分布点㊂2009年3月自昆明开始沿三江并流的峡谷,从南往北详细记录了横断山区云南松和高山松的种群开花物候特征,并应用GPS对高山松和云南松种群分布的经纬度以及海拔进行了精确的定位㊂1.3　云南松和高山松地理分布图的绘制在Diva⁃Gis网站提供的链接(http://biogeo.berkeley. edu/bgm/gdate.php)上下载中国行政区划图层作为底图,并结合中国三级以上河流水系底图,将收集到的高山松和云南松种群分布的地理坐标导入到ArcGIS软件中,生成高山松和云南松隔离地带的地理分布图㊂结合Google earth软件,将物种分布的地理信息与地表地貌信息联系起来,分析高山松和云南松之间可能的地理隔离机制㊂1.4　气象数据及其分析气象数据来源于世界气候数据库(Hijmans等, 2005;/)㊂该数据库对1950-2000年来自世界各地气象站的气候信息通过采用空间插值法生成全球气象数据,因而满足本研究需要㊂选择7个对物种开花物候有重要影响的温度变量,其中包括年均温㊁最暖月均温㊁月均温的年变幅㊁极端高温㊁最冷月均温㊁极端低温㊁年日照时数㊂对所调查的物种分布地点的气象因子采用欧式距离,应用SPSS16.0软件用最短距离法进行聚类分析㊂表1　高山松和云南松的形态学比较(Wang和Szmidt,1994) Table1　Comparative morphology between P.densata and P.yunnanensis特征Character种名Species高山松Pinus densata云南松Pinus yunnanensis 针叶数2-33(稀2)针叶长(cm)6-1510-30针叶直径(mm) 1.0-1.5 1.0-1.2球果长(cm)5-65-11球果直径(cm)4-54-7种子长(mm)4-64-5鳞盾隆起扁平小枝亮黄褐色淡红褐色072 植物分类与资源学报 第33卷2　研究结果2.1　云南松和高山松的地理分布及其界定标本记录和沿路踏查表明高山松分布在西藏东南部㊁四川西部㊁云南西北部和青海南部海拔2700~4200m 的高山地带,在此区域高山松分布不连续;而云南松分布在云南㊁贵州㊁广西西北部和四川西部海拔600~3100m 的高原地带㊂在地理上两物种的临界区位于云南的中甸(图1)㊂整个横断山区的地势形成西北向东南逐级下降的趋势㊂分为三种不同的地貌(图2)㊂昌都-江达-甘孜-马尔康一线以北,高原海拔在4000m 左右,而谷底多在3400~3500m 间,相对高差仅300~500m,成为丘原地貌㊂往南到乡城-稻城-九龙一线,谷底海拔下降到2500~2600m,山地普遍在4200~4500m,相对高差2000m,成为高山河谷地貌㊂再往南到中甸-木里一线,高原面再次下降到3000~3400m,河流深切而密集,相对高差在2000~3000m,成为高山峡谷地貌㊂该区域的典型地貌为植被的分化提供了广阔的空间环境㊂高山松多分布于高山河谷以及高山峡谷地域,云南松则分布在更南端,两物种分别享有不同的生态环境(图1,2)㊂图1　高山松和云南松的隔离带种群地理分布图Fig.1　The geographical distribution of P.densata and P.yunnanensis in isolationregion图2　横断山区三种地表地貌的分界线Fig.2　The boundary of three cross⁃sectional surface topography of the Hengduan Mountains1723期 刘永良等:同倍杂交种高山松与亲本种云南松的地理隔离研究 2.2　高山松和云南松的开花物候特征云南松的开花物候早于高山松,云南松三月下旬就进入开花散粉期,高山松开花散粉期发生在四月,不同的群体波动较大,理县㊁宝兴和小金三个地点种群的开花物候发生于四月上旬,德钦㊁康定㊁马尔康高山松群体四月下旬进入开花散粉期(表2)㊂2.3　依据气象因子的聚类分析基于7个生物气象因子变量,聚类结果明显分为两支(图3),几乎所有的云南松群体聚为一支,高山松群体聚为另一支,中甸的群体作为高山松分布区的最南端群体,因而作为高山松和云南松分布的临界区(图2)㊂中甸地处横断山区的两种地貌分界线上(图2),在地表形态上将两物种分离开来㊂3　讨论3.1　高山松和云南松的地理分布及开花物候差异对两物种的分布界定和开花物候的研究有助于了解同倍杂交种高山松和亲本种云南松之间的隔离机制及高山松的杂种形成机制㊂地质历史和气候变化对物种的分布格局有着重要的影响(Li,1952;Florin,1963;Ying,1983;Wen,2001;Xiang 和Soltis,2001)㊂高山松和云南松分布于不同的地理区域,占据着不同的生态环境,地形地貌显著差异,海拔差异剧烈㊂推测数百万年前的表2　高山松和云南松的开花物候时间Table 2　The flowering phenology time of P.densata and P.yunnanensis 物种　Species 地点Location经度(°E)Longitude 纬度(°N)Latitude 海拔(m)Altitude 起始时间Start time结束时间End time　P.yunnanensis 贡山98.6328.0117363月15日3月28日　P.yunnanensis 洱源99.9526.1120853月17日3月30日　P.yunnanensis 剑川99.9126.5421963月20日4月5日　P.yunnanensis 楚雄101.325.0117723月21日4月10日　P.yunnanensis 昆明102.624.9719003月22日4月11日　P.yunnanensis 大理100.1025.4319903月22日4月11日　P.yunnanensis 维西99.3127.1323253月24日4月6日　P.densata 小金102.5031.0224674月2日4月9日　P.densata 宝兴102.8030.7221804月5日4月14日　P.densata 理县毕棚沟103.0031.4021584月7日4月19日　P.densata 理县米亚罗102.8031.6627464月9日4月21日　P.densata 德钦98.8828.4434494月15日4月25日　P.densata 康定101.9030.1929144月18日4月29日　P.densata 马尔康102.2031.9125264月22日5月1日图3　基于调查群体七个气象因子聚类图气象因子包括年均温㊁最暖月均温㊁月均温的年变幅㊁极端高温㊁最冷月均温㊁极端低温和年日照时数Fig.3　UPGMA clustering of the surveyed populations based on seven bio⁃climate variablesThe bio⁃climate variables including annual mean temperature,annual range of monthly mean temperature,mean temperature for the warmest month,mean temperature for the coldest month,extreme highest temperatures,extreme lowest temperature and annual sunshine hours272 植物分类与资源学报 第33卷地质历史包括海平面改变㊁地壳变动和板块构造等,对高山松和云南松的地理分布现状产生了深远的影响㊂有相似的研究表明,影响日本琉球群岛上植物区系分化(Nakamura等,2009)的因素包括地质历史和当前的生态环境因素(Linder,2001;Fattorini和Fowles,2005;Haus⁃dorf和Hennig,2005)㊂开花物候主要与物种本身㊁开花前的积温㊁花期内的积温等因素有关(沈熙环等,1985),处于异质生态环境中的高山松和云南松群体在建立和发展中经历了不同的生态过程,在长期的生物群体与环境的相互作用过程中,受到强烈选择压力的作用,造成了开花物候的差异㊂3.2　高山松和云南松可能的地理隔离机制在不同的地理区域内,气候条件和土壤条件差异很大,长期以来两物种均得到了适应性的进化㊂同倍杂交种高山松与其亲本种云南松分布在不同的区域,由于地理因素的原因高山松和云南松分布区内的温度相关变量差异较大,温度作为影响开花物候的主要因素,云南松开花物候较早,高山松开花物候较晚,在繁殖期内造成两物种之间的花期不遇,产生了物候隔离,两物种分别在不同的时间段内开花散粉并繁殖后代㊂高山松和云南松的临界区域中甸恰好位于三江并流区域,该区域做作为横断山区的一个瓶颈,云南高原过渡到青藏高原,独特的地表特征形成了烟囱式的地貌,在垂直水平上该区海拔急剧上升,生境发生显著的转变(Chang,1981)㊂临界区内高大的山系等机械隔离在空间上影响了花粉和种子的散播,物种之间不能进行有效的基因交流㊂这两方面的原因共同造成了同倍杂交种高山松和亲本种云南松时间和空间上的合子前隔离(Dobzhan⁃sky,1937;Butlin,1987;Turelli等,2001;Ma⁃clean和Greig,2008;Widmer等,2008)㊂3.3　高山松的适应性进化来自群体遗传学证据表明高山松群体具有较高的遗传多样性,不同的群体具有不同的遗传组成和亲本交配组合㊂在临近云南松分布区的中甸高山松群体中,母系遗传的线粒体单倍型组成中云南松类型占62%㊁油松类型占38%,父系遗传的叶绿体单倍型组成中有68.8%的云南松类型(Song等,2002;Song等,2003)㊂说明高山松成种后并没有立刻与油松和云南松形成完全的生殖隔离,而是存在复杂的甚至是反复的回交过程,在高山松和云南松临界区域内两物种之间存在着基因渐渗㊂高山松成种过程中合子前隔离作用可能较大,但是临界区可能是两物种间的渐渗杂交成功的一个有效的桥梁(Anderson,1949)㊂高山松的形态解剖学性状介于两亲本种之间(Fu等,1999),物种性状研究表明高山松在其亲本种不能正常生长繁殖的高海拔生境中具有正常的生殖能力(Mao等,2009),此外高山松与其亲本种之间还产生了严格的生态分化,有研究表明,高山松在高海拔生境中,在长期的进化历史中固定下来了一系列极端生理学特征,如较快的种苗生长速度,更高的水分利用效率和极端耐干旱能力(Ma等,2010)㊂高山松在进化过程中一些基因位点是经过选择而保留下来的(Ma 等,2006)㊂一系列超亲分离性状的出现经过自然选择作用和适应性进化,在有利的生境中固定下来,为物种形成并适应新的生态环境提供了必要的生物学基础,同时也避免了其与亲本种之间的生存竞争(Rieseberg和Carney,1998;Buerkle 等,2000;Rieseberg等,2003;Seehausen,2004)㊂综上所述,在长期的生态选择作用下,同倍杂交种高山松与其亲本种相比,占据了其亲本种不能存活的新生态位,开花物候较迟以及一些列的种实性状㊁生理学和形态学特征均与其高海拔生境相适应,二者之间产生了显著的地理隔离㊂高山松占据适合其生长繁殖的生态位,生长在其亲本云南松和油松均不能生长的高海拔㊁高寒地域,广泛分布开来㊂此外,本研究是依据形态和气象特征识别物种并推定物种分布界限的,是否可作为遗传学水平上的界限需要进一步的深入研究㊂〔参　考　文　献〕云南植被编写组,1987.云南植被[M].北京:科学出版社王宝田,潘志刚,1963.西南高山林区森林综合考察报告[M].中国林业科学院,326 327四川植被协作组,1980.四川植被[M].成都:四川人民出版社,151 156吴玉虎,2008.青藏高原维管植物及其生态地理分布[M].北京:科学出版社,80 81张荣祖,1997.横断山区自然地理[M].北京:科学出版社,3723期 刘永良等:同倍杂交种高山松与亲本种云南松的地理隔离研究 83 84陈伟烈,1988.西藏植被[M].北京:科学出版社,121 127 Anderson E,1949.Introgressive Hybridization[M].New York: John Wiley and SonsBuerkle CA,Morris RJ,Asmussen MA et al.,2000.The likelihood of homoploid hybrid speciation[J].Heredity,84:441 451 Butlin R,1987.Speciation by reinforcement[J].Trends in Ecology and Evolution,2:8 13Chang D,1981.The vegetation zonation of the Tibetan Plateau[J].Mountain Research and 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中国畜牧兽医2019,4&(2) $48-556China Anim al Husbandry &Veterinary Medicine热溶血曼氏杆菌的分离鉴定及部分生物学韩小丽，任静静，杨铭伟，朱玲，张锐，剡根强"(石河子大学动物科技学院，石河子832000)摘要：为探明一起肉牛运输热的病原及生物学特性，本研究无菌采集病死牛心血、肺脏、肝脏和脾脏，对其进行细菌分离、生化试验和P C R鉴定，并对分离株进行毒力基因检测、致病性研究。

结果显示，7株分离菌均为革兰氐阴性短杆菌，具有微弱的-溶血，瑞氐染色可见两极浓染及明显的荚膜。

生化试验结果显示，分离菌能发酵葡萄糖、麦芽糖、阿拉伯糖、甘、甘露糖、木糖等碳水化合物，不发酵脲酶、M R-V P和吲哚，产生少量酸而不产气，结果符合溶血曼氐杆菌生化特性。

P C R鉴定均为荚膜血清A2型，分离菌均含有四型菌毛相关基因3/A、参与复制相关基因dnaiV、白细胞介素相关基因L3C 3种毒力基因。

分离菌对小鼠的L D5〇值在107_83*108_50C F U/m L之间，不同菌株间小鼠L D5〇值存在一定差异，但差异不明显。

结果表明，引起该批肉牛运输热的病原为携带毒力基因的荚膜血清A2型溶血曼氐杆菌，本研究结果为进一步研究溶血曼氐杆菌的致病机制提供参考。

关键词：肉牛；溶血性曼氐杆菌；鉴定；生物学特性中图分类号：S852. 61+2文献标识码：AD o i：10. 16431/j. cnki. 1671-7236. 2019. 02. 026 开放科学（资源服务）标识码（O S I+):Isolation，Identification and Partial Biological Characteristics ofM a n n h e im ia h a e m o ly t ic a in Shipping Fever of Beef CattleH A N Xiaoli，RENJingjing，Y A N G Mingwei，Z H U Ling，Z H A N G Rui，Y A N Genqiang"(.College〇/Anim al Science and Technology，S hihizi U niversity，Shihezi 832000，China)Abstract： In order to identify the pathogens and biological characteristics of shipping fever of beefcattle.The bacteria of blood (heart),lung,liver and spleen were collected and isolated,biochemi­cal tested,and P C R identified,the virulence gene detection and pathogenicity o studied.The results showed that 7 isolates were Gram-negative brevibacterium with weak p-he-molysis ,Wright?s staining result showed that there was two-pole dense staining and obvious cap­sule. The biochemical test r esults showed that the isolated bacteria could ferment carbohydratessuch as glucose,maltose,arabinose,mannitol,mannose,xylose etc. and do not ferment urease，M R-V P and indole. I t could utilize carbohydrates such as glucose to produce a small amount ofacid without gas production ,and the results were consistent with the biochemical characteristicsof Mannheimia haemolytica.The isolates were capsular serum A2 type by P C R,which containedthree virulence genes of the type — fimbriae-related gene pt/A.，t he copy-related gene d n a N，andthe interleukin related g ene L ktC. The LD50 values of the bacteria in mice ranged from 107 83 to108 50C F U/mL,therewere some difference of L D50 values in different strains.The results showedthat the pathogen which caused the shipping fever in the batch of b收稿日期：2018-06-12基金项目：国家科技支撑计划项目"012B A D43B02)作者筒介：韩小丽（1994-)，女，新疆乌苏人，硕士，研究方向：动物传染病预防与防治，E-mail: 964242597@qq. com"通信作者：剡根强a958-)，男，甘肃西和人，教授，博士生导师，研究方向：预防兽医学，E-mail:ygq58®2期 韩小丽等：致肉牛运输热溶血曼氏杆菌的分离鉴定及部分生物学特性研究haemolytica with c apsular serum A2 type,which provided a reference for further study of the pathogenic mechanism of Mannheimia haemolytica%Key words：beef；Mannheimia haemolytica identification；biological characteristics溶血曼氏杆菌（M a n n h#miahaemoZ；y#ca ,M. haemolytica)原名溶血性巴氏杆菌（PasewreZZa haemoZyica),栖息在鼻咽部与宿主保持一种共生的关系[12]，是引起运输热（shipping fever)即牛呼吸 道疾病综合征（bovine respiratory disease complex, B R D C)的病原之一，具有全球分布性）]。

专利名称：Isolation and structure of the human cancer cell growth inhibitory cyclic octapeptidesphakellistatin 10 and 11发明人：George R. Pettit,Rui Tan申请号：US08/360239申请日：19941220公开号：US05801222A公开日：19980901专利内容由知识产权出版社提供摘要：Two new compounds which may be useful in the treatment of one or more neoplastic diseases through chemotherapy have been isolated from the Western Pacific Ocean marine sponge Phakellia sp. The cyclic octapeptides phakellistatin 10 and phakellistatin 11 are disclosed herein.< P>As the included data demonstrate, phakellistatin 10 attained an LC. sub.50 of less than 10.sup.-5 M per milliliter against the MDA-MB-435 breast cancer cell line. It also achieved total growth inhibition for two breast cancers and two CNS cancer cell lines at a concentration of less than 10.sup.-6 M per milliliter. The included data for phakellistatin 11 shows a moderately higher level of in vitro activity as shown by an LC. sub.50 of less than 10.sup.-5 M per milliliter against the MDA-MB-435 breast cancer cell line and five other cell lines. It also achieved total growth inhibition for two breast cancers, one CNS cancer, one Ovarian cancer, and one non-small cell lung cancer cell lines at a concentration of less than 10.sup.-7 M per milliliter.申请人：ARIZONA BOARD OF REGENTS ACTING ON BEHALF OF ARIZONA STATE UNIVERSITY代理人：Richard R. Mybeck 更多信息请下载全文后查看。

Induction of apoptosis and autophagy in human glioblastoma cells byN -methylflindersine ： insights into regulatory role of ERK pathway *LI Siyuan 1， LUO Yuyou 1， LUO Xiongming 3， WANG Zhongyu 1， CHEN Huitong 1，FAN Dong 1， YUAN Xingyi 1， CHEN Le 1， TANG Pei 1， LIU Jing 1△，WANG Zongming 2△， WANG Xin 1△（1Department of Human Anatomy ， Histology and Embryology ， School of Basic Medical Sciences ， Guangdong Pharmaceuti⁃cal University ， Guangzhou 510006， China ； 2Pituitary Tumor Center ， Department of Neurosurgery ， the First Affiliated Hos⁃pital ， Sun Yat -sen University ， Guangzhou 510062， China ； 3School of Life Sciences and Biopharmaceutics ， GuangdongProvincial Key Laboratory of Pharmaceutical Bioactive Substances ， Guangdong Pharmaceutical University ，Guangzhou 510006， China ）［ABSTRACT ］ AIM ： Mangrove -associated plants are known for producing natural compounds with antitumor ac⁃tivity. Despite the potential therapeutic value of these compunds ， the molecular mechanisms underlying their antitumor ef⁃fects remain unclear. This study aimed to investigate the antitumor properties of N -methylflindersine ， an alkaloid derived from the mangrove -associated plant ， Micromelum falcatum （Lour.） Tan.， and its effects on U87 human glioblastoma cells. METHODS ： We identified and isolated 15 compounds from the stem bark of Micromelum falcatum . Among these ， we screened N -methylflindersine for its potential inhibitory effects on U87 cell growth. Various assays ， including wound healing ， Hoechst 33342/PI staining ， and protein expression analysis ， were conducted to investigate the compound's im⁃pact on cell migration ， apoptosis ， and autophagy -related proteins. RESULTS ： Within 24 h ， N -methylflindersine demon⁃strated the ability to reduce U87 cell migration and increase the apoptotic U87 cell population. Furthermore ， it downregu⁃lated the anti -apoptosis protein Bcl -2 expression ， upregulated the pro -apoptosis protein Bax expression ， and elevated the ratio of autophagy -related protein LC3-II/LC3-I in U87 cells. Additionally ， the ERK signaling pathway was found to be down -regulated following N -methylflindersine treatment. CONCLUSION ： N -methylflindersine appears to induce bothapoptosis and autophagic cell death in U87 cells ， resulting in reduced cell growth. This effect seems to be associated with the downregulation of the ERK signaling pathway.［KEY WORDS ］ mangrove -associated plants ； N -methylflindersine ； glioblastoma［CLC number ］ R739.4； R966； R363.2 ［Document code ］ A doi ： 10.3969/j.issn.1000-4718.2024.04.003Oceans are abundant repositories of natural com⁃pounds. Mangroves are unique marine ecosystems dis⁃tributed along the coasts of tropical and subtropical re⁃gions ［1-2］. In addition to widely researched true man⁃groves and mangrove -associated fungi ， mangrove -asso⁃ciated plants growing in the intertidal zone have shown great bioactivity and beneficial application ［3-4］. For in⁃stance ， the aqueous seed extract of Derris trifoliataLour （a common mangrove liana ） has a significantly high 2，2-diphenyl -1-picrylhydrazyl （DPPH ） scaveng⁃ing activity ， multiple antibacterial activities ， and a moderate inhibitory effect on A549 cells ［5］. The leaf wa⁃ter extract of Barringtonia racemosa （another mangrove -associated plant ） modifies the cell cycle and triggers apoptosis in Caco -2 cells ［6］. Some mangrove -associatedplants were formerly classified as true mangroves ； how⁃［文章编号］ 1000-4718（2024）04-0594-08［Received date ］ 2023-11-06 ［Accepted date ］ 2023-12-06* ［Foundation item ］ Supported by the National Natural Science Foundation of China for Young Scholars （No. 81802678）， the Guangdong Natural Science Foundation Project （No. 2018A030310107）， the Guangzhou Basic and Applied Basic Research Founda⁃tion （No. 202102021116； No. 202201010307； No. 202201010299）， and the Guangdong Province Administration of Traditional Chi⁃nese Medicine Project （No. 20231210）.△Corresponding author WANG Xin E -mail ： wangxin@ ； LIU Jing E -mail ： liujing@ ； WANG ZongmingE -mail ： wangzm23@··594ever，they have received more precise descriptions in recent years， including distinctions in leaf features and salt tolerance［7-8］.Some mangrove-associated plant ex⁃tracts from different genera exhibit antitumor activi⁃ty［9-11］.This indicates that these plants may be attrac⁃tive sources of antitumor medicines.Glioblastomas are the most aggressive brain tu⁃mors owing to their frequent occurrence and invasive characteristics within the central nervous system （CNS）［12］.This rapidly developing malignancy has a World Health Organization （WHO） grade 4 CNS tumor， with a poor prognosis that affects the elderly and chil⁃dren［13］.Although surgery，high-dose radiation，and chemotherapy help alleviate patient symptoms，com⁃plete glioblastoma control remains a serious problem that must be conquered globally［12］. Therefore， more in⁃novative antineoplastic medicines are urgently re⁃quired.Xyloketal B is a constituent of the mangrove fungus Xylaria sp.（No.2508）that inhibits U251 cells by attenuating the MEK/ERK and PI3K/Akt path⁃ways［14］. Mangrove-associated compounds may serve as weapons against glioblastomas.In this study，15 compounds were extracted from the stem bark of Micromelum falcatum（a mangrove-as⁃sociated plant）， including ten coumarins 1-10， four al⁃kaloids 11-15， and a new compound （compound 13）. Compound 13 （N-methylflindersine）was the focus［15］and exhibited an inhibitory effect against U87 cells. This study explored its antitumor properties and poten⁃tial mechanisms.MATERIALS AND METHODS1　Compound extraction and isolation1.1　General　All nuclear magnetic resonance （NMR）experiments were performed using a Bruker DRX-500 spectrometer.1H NMR was conducted at 500 MHz，and 13C-NMR was conducted at 125 MHz，equipped with an inverse-detection 5 mm probe operat⁃ing at room temperature. SiMe4 was utilized as the inter⁃nal standard and samples were dissolved in deuterated solvent to test the NMR spectra.Optical rotation was measured using a Polaptronic-HNQW5 high-resolution polarimeter，and the infrared （IR）spectrum was ob⁃tained using a Bruker VECTOR22 infrared spectropho⁃tometer. High-resolution electron ionization mass spec⁃trometry （HREI-MS）and electrospray ionization-mass spectrometry （ESI-MS）were performed using Thermo MAT95XP and API2000 LC/MS/MS mass spectrome⁃ters （Applied Biosystems），respectively.Silica gel served as the medium for the column and thin-layer chromatography （TLC） plates. The TLC plates were vi⁃sualized by applying a mixed solvent consisting of 20% sulfuric acid and 80% ethanol， followed by heating. A Waters 600 high-performance liquid chromatography （HPLC）system equipped with a Waters 996 photodi⁃ode array detector was used for HPLC analysis and semi-preparative reversed-phase （RP）-HPLC （Wa⁃ters）with an octadecylsilane （ODS）column （YMC-Pack ODS-5-A， 250×10 mm i.d.， 5 μm， YMC） was performed using the CH3OH/H2O solvent system as elu⁃ents.Column chromatography was performed using a Sephadex LH-20 column （Pharmacia） as the stationary phase.1.2　Plant material　In October 2010，M. falcatum （Lour.）Tan.were collected from Hainan Province，China.Prof.Jun Wu from the South China Sea Insti⁃tute of Oceanology， Chinese Academy of Sciences veri⁃fied the identity and deposited a voucher specimen in the herbarium of the South China Sea Institute of Ocean⁃ology.1.3　Extraction and isolation　Air-dried M.falca⁃tum（lor）Tan.（11.5 kg）was extracted three times with 95% EtOH （50 L）and 50% EtOH （50 L）.The organic solvent was pooled and evaporated under re⁃duced pressure.The residue was dissolved in2.5 L H2O and successively extracted with hexane and ethyl acetate （EtOAc； three times each） to yield 58 g of hex⁃ane extract and 117 g of EtOAc extract. The EtOAc ex⁃tract was fractionated on silica gel （1 755 g， 200~300 mesh）using solvents with ascending polarity：10%~ 70% acetone in n-hexane，followed by 5%~100% MeOH in CHCl3，resulting in 122 fractions.Frs.9-12 （3.70 g，eluted with n-hexane-acetone 90∶10）was subjected to silica gel chromatography with a mixture of chloroform and acetone （40∶1） and further purified on Sephadex LH-20 using methanol （MeOH）to yield 12.4 mg of 12， 21.8 mg of 13 and 7.5 mg of 14. Frs. 33-36 （3.20 g，eluted with n-hexane-acetone 70∶30）was combined and chromatographed on silica gel with chloroform-acetone （15∶1） to afford Frs. 36a-d. Com⁃··595pound 36b was further purified by chromatography on a Sephadex LH-20 column using CHCl3/MeOH to afford compound 9 （151 mg）.Frs.40-43 （2.77 g，eluted with a mixture of n-hexane and acetone in a ratio of 70∶30）were chromatographed on a silica gel with chloro⁃form-acetone （7∶1） to afford 426 mg of 5. Frs. 45-47 （4.17 g， eluted with a mixture of n-hexane and acetone in a ratio of 70∶30） were fractionated on silica gel with chloroform-acetone （9∶1 and 6∶1）to afford fraction 47a-d. Compound 47a was purified using chromatogra⁃phy on a Sephadex LH-20 column to afford 20.7 mg of1. Compound 47b was separated by chromatography ona Sephadex LH-20 column and semi-preparative HPLC to afford 10.5 mg of 4 and 11.2 mg of 6.Frs.55-60 （3.35 g，eluted with n-hexane-acetone 60∶40）was fractionated on a silica gel using chloroform-acetone （8∶2） to afford fraction 60a-b. Compound 60a was further purified by chromatography on a Sephadex LH-20 col⁃umn to yield compound 8 （20.9 mg）， which was crys⁃tallized from CHCl3. Compound 60b was purified using semi-preparative HPLC with MeOH/H2O as the eluent，yielding compound 10 （12.5 mg）.Frs.63-65 （4.36 g， eluted with n-hexane-acetone 50∶50） was separated on silica gel using chloroform-acetone （5∶1）to obtain 15.2 mg of 7） and further purified by semi-preparative HPLC （MeOH/H2O） to yield 11.0 mg 2 and 8.2 mg 3. Frs. 68-71 （3.65 g， eluted with n-hexane-acetone 50：50）was separated on silica gel （200~300 mesh）with chloroform-acetone （3∶1） and purified using Sephadex LH-20 and semi-preparative HPLC to yield 42.5 mg of 11 and 12.6 mg of 15.2　Cell cultureU87， U251 and 4T1 cells were maintained in Dul⁃becco's modified Eagle's medium （DMEM）（Gibco，USA） supplemented with 10% （v/v） fetal bovine serum （FBS）and 1% penicillin/streptomycin.Cells were in⁃cubated in a humidified atmosphere containing 5% CO2 at 37 ℃， and CT-26 was cultured in Roswell Park Me⁃morial Institute （RPMI）1640 medium under similar conditions.3　Cell viability assayCell viability was assessed using the Cell Counting Kit-8 （CCK-8）assay （Biosharp，China）.Cells （1×104） were cultured in a 96-well plate with 100 μL medi⁃um. After 24 h， the cells were treated with N-methyl⁃flindersine for another 24 h.Subsequently，10 μL of CCK-8 reagent was added，and incubated for 1 h at 37 ℃，and the absorbance was measured at 450 nm using a microplate reader （Bio-Rad， USA） to calculate the IC50.4　Wound healing assayU87 cells （1×104） were seeded in 6-well plates， al⁃lowed to adhere overnight， and scratched with a pipette tip.The cells were washed with PBS，treated with N-methylflindersine at IC50concentration，and incubated for 24 h. Images were captured at 0 and 24 h after treat⁃ment，and migration was analyzed based on scratch width changes in three different fields per well.5　Hoechst 33342/propidium iodide （PI）fluores⁃cence stainingApoptosis was assessed by Hoechst 33342/PI fluo⁃rescence staining. U87 cells （5×105） were seeded into 24-well culture plates and cultured overnight. The cells were incubated with the IC50of N-methylflindersine in culture medium for 24 h， followed by the addition of a mixture of Hoechst 33342 and PI （Biosharp， China），and stained at 37 ℃ for 30 min. The cellular morpholo⁃gy was examined using a fluorescence microscope （Lei⁃ca，Germany）.Three different fields were analyzed within each well.6　Western blot assayU87 cells （2×106）were treated with N-methyl⁃flindersine at IC50for 24 h，lysed，and quantified. Equal amounts of protein were separated by sodium do⁃decyl sulfate-polyacrylamide gel electrophoresis， trans⁃ferred to polyvinylidene difluoride （PVDF）mem⁃branes， probed with primary and secondary antibodies，and visualized using chemiluminescence. The band in⁃tensities were then quantified.7　Statistical analysisStatistical analyses were performed using Graph⁃Pad Prism 5.0 software. The outcomes are presented as mean ± standard deviation （mean±SD）.Differences between pairs of datasets were evaluated for statistical significance using the Student's t-test.A P-value less than 0.05 was considered to be statistically significant. RESULTS1　N-methylflindersine inhibited U87 cell viability Initially， the dose-dependent effects of compounds··5961-15 on the viability of human glioblastoma U87 cell line were investigated using a CCK -8 assay. The cells were exposed to increasing concentrations of the com⁃pounds for 24 h ， and the viability of the cells was sup⁃pressed in an N -methylflindersine dose -dependent man⁃ner （Figure 1）. The IC 50 values of the compounds against U87 cells were determined （Table 1）. Nota⁃bly ， N -methylflindersine only affected U87 cells with an IC 50 value of 17.372±0.434 μmol/L compared to the much lower IC 50 values of another human glioblastoma cell line ， U251， the mouse breast cancer cell line ， 4T1， and the mouse colon cancer cell line ， CT -26.This indicated that U87 cells exhibited the highest sen⁃sitivity to N -methylflindersine treatment （Table 2）.2　N -methylflindersine inhibited migration of U87cellsCancer cells can metastasize to distant parts of thebody ， which is a characteristic of tumors. We conducted a wound healing assay to test whether N -methylflinder⁃sine treatment could curb the migration of U87 cells.Inhibition of wound closure was observed after N -methyl⁃flindersine treatment in the micrographs （Figure 2）.3　N -methylflindersine induced apoptosis of U87 cellsU87 cells were investigated using Hoechst 33342and PI fluorescence dyes to track nuclei in mid -to -late apoptosis. Activation of apoptosis was observed in U87 cells after N -methylflindersine treatment （Figure 3A ）. A western blot assay was used to evaluate the expres⁃sion of the survival protein Bcl -2 and the apoptosis -in⁃ducing protein Bax to investigate whether N -methyl⁃flindersine -induced cytotoxicity in U87 cells involved modulation of pro - and anti -apoptotic genes. There wasa substantial decrease in Bcl -2 protein expression ， cou⁃pled with a notable increase in Bax protein levels fol⁃lowing N -methylflindersine treatment （Figure 3B ）.4　N -methylflindersine induced autophagy ofU87Figure 1.　The structure of N -methylflindersine.Figure 2.　N -methylflindersine inhibited the migration of U87cells （detected by wound healing assay ）. The area within the white dashed line shows the cells that mi⁃grated in 24 h. Scale bar=400 μm.Table 1.　The IC 50 of 15 kinds of compounds for U87 cells Number 123456789101112131415NameMicromeloside A Micromeloside CMicromeloside D Murrangatin Micromelin MinumicrolinHydramicromelin A MurracarpinMicrominutinin Microfalcatin Isovalerate Micrometam B 3-Hydroxy -1-methyl -3-（2-oxopropyl ）quinoline -2，4（1H ，3H ）-dioneN -MethylflindersineN -Methylswietenidine -BCatunaregin IC 50 （μmol/L ）>200>200>200>200>200>200>200>200>200>200>200>20017.372±0.434>200>200Table 2.　The IC 50 of N -methylflindersine for 4 cell lines Cell lines U874T1U251CT -26IC 50 of N -methylflindersine （μmol/L ）17.372±0.434100~200>200>200··597cellsAutophagic cell death is an important mechanism incancer treatment through drug intervention. N -methyl⁃flindersine significantly increased the ratio of autopha⁃gy -related protein LC3B （LC3-II to LC3-I ratio ） in U87 cells according to Western blot analysis （Figure 4）.5　N -methylflindersine suppressed ERK pathway inU87 cellsMitogen -activated protein kinases （MAPKs ） in⁃cluding ERK mediate cell migration and apoptosis. Western blot analysis showed that the phosphorylated ERK （p -ERK ）/ERK ratio significantly decreased at theIC 50 of N -methylflindersine relative to that of the corre⁃sponding control （Figure 5）.DISCUSSIONMangrove -associated plants are vital sources ofnatural products. In recent years ， many compounds ex⁃tracted from mangrove -associated plants exhibit antitu⁃mor activities ［4］. Fifteen compounds were successfullyextracted from M. falcatum using advanced natural compound extraction and separation methods. N -methyl⁃flindersine alkaloid exhibits antitumor activity in U87cells and was chosen for further research. N -methyl⁃flindersine might attenuate the migration ability of U87Figure 3.　N -methylflindersine induced apoptosis of U87 cells. A ： the fluorescence staining results of U87 cells with Hoechst33342 and PI marked after N -methylflindersine treatment （scale bar=100 or 50 μm ）； B ： the Bax and Bcl -2 protein expres⁃sion levels in U87 cells. Mean±SD. n =3. **P <0.01 vscontrol group.Figure 4.　N -methylflindersine induced autophagy of U87cells. The LC3-I and LC3-II protein levels in U87 cells were detected by Western blot. Mean±SD. n =3. *P <0.05 vs control group.··598cells. Combined with the experimental results of CCK -8， N -methylflindersine was hypothesized to inhibit U87 cells ， although the underlying mechanisms must be elu⁃cidated.The most extensively investigated mechanism ofcell death is regulated cell death （RCD ）， which relies on dedicated molecular machinery that can be modulat⁃ed by pharmacological or genetic interventions. It in⁃volves apoptosis and autophagy -dependent cell death ， allowing the body to control the balance between cell growth and death. The effectors of these two pathways are elevated in processes involving medications to pre⁃vent and treat glioblastoma［16］.Apoptosis is induced by intracellular and extracel⁃lular stimulation and is mediated by mitochondrial and death receptor -mediated pathways. The B -cell lympho⁃ma 2 （Bcl -2） family of proteins contains pro -apoptotic （such as Bax ） and anti -apoptotic （such as Bcl -2） mem⁃bers and is needed to regulate and execute intrinsic apoptosis. Over the last two decades ， targeting Bcl -2 family members has aided in cancer therapeutic phar⁃macological treatments［17］. This study found that N -methylflindersine enhanced the apoptotic rate in U87cells. Simultaneously ， Bax protein expression in⁃creased while Bcl -2 protein expression decreased. Thisimplies that N -methylflindersine may trigger U87 cells ’ intrinsic apoptotic mechanism.Subsequently ， we focused on type II RCD and au⁃tophagy -dependent cell death in U87 cells. In cancer biology ， the intracellular self -degradation pathway known as autophagy is frequently viewed as a double -edged sword. It plays two roles in tumor growth and maintains a balance between cytoprotection and cyto⁃static activity ［18］. LC3-I may change to LC3-II during autophagosome formation ， mediating membrane closure and the fusion of autophagosomes with lysosomes ［19］. A crucial step in autophagy is the accumulation of LC3-II. In this study ， N -methylflindersine treatment of U87 cells led to upregulation of the LC3-II protein ， even with an increased ratio of LC3-II to LC3-I ， demonstrat⁃ing more pronounced autophagy activation. This is in line with recent studies hypothesizing that autophagy functions as a tumor suppressor in gliomas ［19-20］.The ERK signaling pathway controls a variety ofbiological processes in different tumor types ， such as cell proliferation ， apoptosis ， migration ， and inva⁃sion ［21-22］. The ERK pathway can facilitate and inhibit apoptosis in diverse contexts ， and hyperactive ERK sig⁃naling is frequently linked to anti -apoptotic effects ［23］. For example ， the activation of ERK ， along with its up⁃stream signals c -Raf and MEK in Helicobacter pylori -in⁃fected GES -1 cells can prevent H. pylori -induced apop⁃tosis ［24］. Conversely ， suppression of the MEK/ERK sig⁃naling pathway activates the intrinsic apoptotic pathwayin A549 cells ［25］. Niclosamide inhibits U87-MG cell growth by inducing autophagic responses and blocking the pro -survival signal transduction pathway MAPK/ERK ［26］. Although ERK modifications are congruent with changes in autophagy levels ， it appears that theERK pathway is also associated with autophagy -depen⁃dent cell death ［27-28］. Given that the ratio of LC3-II/LC3-I significantly differed between the two groups ， we be⁃lieve that autophagy in U87 cells was hyperactive and caused cell death. Changes in autophagic flow may re⁃quire additional confirmation. In this study ， we found that N -methylflindersine led to downregulation of the ERK signaling pathway in U87 cells. Therefore ， ERKmay be a target of N -methylrutin in glioblastomas ，al⁃Figure 5.　N -methylflindersine suppressed the ERK path⁃way in U87 cells. The p -ERK and ERK protein lev⁃els in U87 cells were detected by Western blot.Mean±SD. n =3. **P <0.01 vs control group.··599though the specific mechanism of action requires fur⁃ther verification.In addition， the CCK-8 assay showed that N-meth⁃ylflindersine only had inhibitory effects on U87 cells，which may be owing to the different inhibitory effects of the same compound on different tumor cells caused by multiple factors，including dose-dependent responses，structural variations， and molecular targets. For exam⁃ple，a study on horsetail ferns found that hexane and chloroform extracts exhibited dose-dependent variations in anticancer activity against human hepatocarcinoma cells，with higher concentrations inducing significant antioxidant and apoptotic effects［29］.Chalcones are a class of compounds with diverse biological activities，including anticancer activities by inhibiting various mo⁃lecular targets， indicating that structural variations can affect their potency［30］. 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Molecules， 2021， 26（4）：833.（责任编辑：余小慧，罗森）N-甲基芸香碱通过调控ERK通路诱导人胶质母细胞瘤细胞凋亡和自噬*黎思源1，罗宇悠1，罗雄明3，王仲宇1，陈晖彤1，樊东1，袁星怡1，陈乐1，唐佩1，刘靖1△，王宗明2△，王欣1△（1广东药科大学基础医学院人体解剖学与组织胚胎学系，广东广州 510006；2中山大学附属第一医院神经外科垂体瘤中心，广东广州 510062；3广东药科大学生命科学与生物制药学院，药用生物活性物质研究所，广东广州 510006）［摘要］目的：已知红树林相关植物能够产生具有抗肿瘤活性的天然化合物。

重要概念解释AAbundance (mRNA 丰度)：指每个细胞中mRNA分子的数目。

Abundant mRNA(高丰度mRNA)：由少量不同种类mRNA组成，每一种在细胞中出现大量拷贝。

Acceptor splicing site (受体剪切位点)：内含子右末端和相邻外显子左末端的边界。

Acentric fragment(无着丝粒片段)：(由打断产生的)染色体无着丝粒片段缺少中心粒，从而在细胞分化中被丢失。

Active site(活性位点)：蛋白质上一个底物结合的有限区域。

Allele(等位基因)：在染色体上占据给定位点基因的不同形式。

Allelic exclusion(等位基因排斥)：形容在特殊淋巴细胞中只有一个等位基因来表达编码的免疫球蛋白质。

Allosteric control(别构调控)：指蛋白质一个位点上的反应能够影响另一个位点活性的能力。

Alu-equivalent family(Alu相当序列基因)：哺乳动物基因组上一组序列，它们与人类Alu 家族相关。

Alu family (Alu家族)：人类基因组中一系列分散的相关序列，每个约300bp长。

每个成员其两端有Alu切割位点(名字的由来)。

α-Amanitin(鹅膏覃碱)：是来自毒蘑菇Amanita phalloides二环八肽，能抑制真核RNA聚合酶，特别是聚合酶II 转录。

Amber codon (琥珀密码子)：核苷酸三联体UAG，引起蛋白质合成终止的三个密码子之一。

Amber mutation (琥珀突变)：指代表蛋白质中氨基酸密码子占据的位点上突变成琥珀密码子的任何DNA改变。

Amber suppressors (琥珀抑制子)：编码tRNA的基因突变使其反密码子被改变，从而能识别UAG密码子和之前的密码子。

Aminoacyl-tRNA (氨酰-tRNA)：是携带氨基酸的转运RNA，共价连接位在氨基酸的NH2基团和tRNA终止碱基的3′或者2′－OH基团上。

分子生物学习题答案第一章绪论Chapter 1 Introduction一名词解释1．人类基因组计划：与曼哈顿原子弹计划和阿波罗登月计划相媲美的美国人类基因组计划（human genome project, HGP)，解读人基因组上的所有基因、24个染色体DNA分子中的碱基序列。

在―人类基因组计划‖中，分为两个阶段：DNA序列图以前的计划和DNA序列图计划。

序列图前计划包括遗传图、物理图、转录图。

2. RFLP (restrict fragment length polymorphism )：A variation from one individual to the next in the number of cutting sites for a given restriction endonuclease in a given genetic locus.3. DNA指纹：基因组中存在着多种重复序列，拷贝数从几个到数十万个，可分为串联重复序列和分散重复序列。

根据个体重复序列拷贝的位置和数目的差异，使用限制性内切酶，获得具有个体特异性的DNA片段。

可以作为亲缘关系或个人身份的鉴定。

4. SNP（single nucleotide polymorphism, 单核苷酸多态性）：在一个群体中，基因组内某一特定核苷酸位置上出现2种或2种以上不同核苷酸的现象，在群体中相应频率为1-2%。

如果低于这个频率，可视为点突变。

二简答1. What is molecular biology？Molecular biology is the subject of gene structure and function at the molecular level.To explain the principle of development, metabolism, heredity and variation, aging at the molecular level. It grew out of the disciplines of genetics and biochemistry.2. Major events in the genetics century第二章核酸、蛋白质结构一选择题：B, E, D, A, A二名词解释1．Transfection：describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer. The term transfection for non-viral methods is most often used in reference to mammalian cells, while the term transformation is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells such as fungi, algae and plants.2．Configuration：The configuration of a molecule is the permanent geometry that results from the spatial arrangement of its bonds. The ability of the same set of atoms to form two or more molecules with different configurations is stereoisomerism.Configuration is distinct from chemical conformation, a shape attainable by bond rotations.3．构象：(Conformation, generally means structural arrangement），指一个分子中不改变共价键结构，仅是单键周围的原子旋转所产生的原子空间排列。

中国细胞生物学学报 Chinese Journal ofCell Biology2021,43(1): 144-151DOI: 10.11844/cjcb.2021.01.0018自噬和铁死亡的相互联系李听李平熊秋宏*(山西大学生物医学研究院，太原030006)摘要 自禮是一个保守的细胞内降解系统，在细胞死亡中起着双重作用，可以为细胞在营养缺乏条件下提供一些必要的营养物质促进细胞存活，但是自噬过度发生会导致细胞内一些正常组 分被降解从而加速细胞死亡。

铁死亡是一种新的细胞死亡调控形式，主要依赖于铁的积累和脂质 过氧化。

铁死亡在细胞形态、生物化学特征和所涉及的调控因子上都与自噬以及其他类型的细胞 死亡方式不同。

然而，最近的研究表明，铁死亡的发生依赖于自噬，并且许多铁死亡调节因子被认 为是潜在的自噬调节因子。

该文主要对自噬和铁死亡相互联系的分子机制进行综述。

关键词自噬;铁死亡;相互联系；分子机制The Crosstalk between Autophagy and FerroptosisLI Xi n,LI Ping,X I O N G Q i u h o n g*(Institutes o f B iomedical Sciences, Shanxi University, Taiyuan 030006, China)Abstract Autophagy and ferroptosis are two forms of R C D(regulated cell death).Autophagy supplies nutrients for the synthesis of essential proteins during starvation,and thus helps to extend cell survival.H o w e v e r, excess autophagy can destroy essential cellular components and lead to cell death.Ferroptosis i s a newly identi­fied R C D whi c h depends on the iron accumulation and lipid peroxidation.T h e mai n features of ferroptosis are significantly distinguished from autophagy in terms of morphology,biochemistry and genetics.Recently,extensive evidences have s h o w n that there i s a crosstalk between these two forms of R C D s.Studies have demonstrated that activation of ferroptosis is dependent on the induction of autophagy.Additionally,m a n y ferroptosis regulators have been identified as potential regulators of autophagy.This review highlights the crosstalk mechanisms between au­tophagy and ferroptosis at the molecular level.K e y w o r d s autophagy;ferroptosis;crosstalk;molecular m e c h a n i s m目前，科学家己经鉴定出12种不同类型的受调 控的细胞死亡(regulated cell death,R C D)方式，其中包 括自噬和铁死亡，不同类型的细胞死亡在形态、生 化、调控蛋白和功能机制方面都是不同的I“自噬’’(autophagy)—词是D E D U V E在1963年时提出的,用来描述通过溶酶体清除和降解细胞内成分(如蛋 白质聚集体和受损的细胞器等)的过程|21。