ap11_frq_macroeconomics_formb

中国瓜菜2024，37（4）：27-35收稿日期：2023-10-10；修回日期：2023-12-20基金项目：烟台市科技计划项目（2022XCZX091）；国家现代农业产业技术体系专项（CARS-23-G11）；重庆市巫山县科技项目（wskjdx-bxm2023004）；国家自然科学基金面上项目（32372737）；西南作物基因资源发掘与利用国家重点实验室开放课题（SKL-KF202224）作者简介：刘佳凤，女，在读硕士研究生，研究方向为番茄抗逆基因的验证。

E-mail ：*******************通信作者：李涛，男，正高级农艺师，研究方向为蔬菜育种及分子生物学。

E-mail ：****************DOI ：10.16861/ki.zggc.202423.0652番茄SlMYB48基因生物信息学及表达分析刘佳凤1，郭晓青2，王桂强3，王虹云4，朱桐1，曹守军4，姚建刚4，张丽莉4，张瑞清4，赵婧1，李涛1，4（1.烟台大学生命与科学学院山东烟台264000 2.烟台市农业技术推广中心山东烟台2654993.招远市张星镇农业综合服务中心山东招远2654034.山东省烟台市农业科学研究院山东烟台264500）摘要：MYB 转录因子是植物转录因子家族中数量最多、用途最广的成员之一，为挖掘更多番茄（Solanum lycopersi-cum ）MYB 转录因子家族成员信息，初步探究其表达模式及功能，以番茄Ailsa Craig 为试材，采用RT-PCR 的方法克隆SlMYB48基因，并对其进行生物信息学及表达、定位分析。

结果表明，番茄SlMYB48基因的开放阅读框（ORF ）长度为708bp ，编码235个氨基酸，在番茄的根中表达量最高，叶中次之。

SlMYB48蛋白含有保守的MYB 结构域，定位于细胞核中，属于不稳定、亲水性蛋白。

对SlMYB48启动子分析，发现其含有大量的逆境响应元件，qRT-PCR 及RNA-seq 数据库分析结果表明，高盐、生物逆境胁迫条件下，SlMYB48基因表达量均随处理时间延长而升高，干旱胁迫条件下表达量下降，推测其可能参与番茄生物及非生物逆境胁迫反应。

第9卷第4期2011年12月生物信息学China Journal of Bioinformatics Vol．9No．4Dec．，2011收稿日期：2010－01－06；修回日期：2010－05－30.基金项目：安徽高校省级自然科学研究重点项目资助（KJ2008A089）.作者简介：詹少华，男，教授，博士，研究方向：生物信息学与分子育种，E －mail ：zhansh@wxc．edu．cn．*通讯作者：林毅，教授，博士生导师，E －mail ：linyiahau@126．com．doi ：10.3969/j．issn．1672－5565．2011．04．08利用VBA 查找核酸数据库DNA 保守序列詹少华1，尹艺林1，蔡永萍2，樊洪泓2，林毅2*（1．皖西学院生物与制药工程学院，六安237012；2．安徽农业大学生命科学学院，合肥230036）摘要：采用VBA 编写了查找核酸数据库保守序列的四个相关程序，“导入DNA 序列”程序可以将Fasta 格式的DNA 序列文本文件存放到Excel Sheet1的A 列中，保留每个序列的Gi 号，删除多余的注释部分；“整理DNA 序列”程序可以将DNA 序列Gi 号存放到A 列中，B 列为对应Gi 号的完整序列；“DNA 随机序列”程序可以产生DNA 随机序列；“发现DNA 保守序列”程序可以将随机序列与下载的DNA 序列比对，查找每一种随机序列的出现频率。

以大豆基因组序列为实例，说明了这些程序的应用方法。

该程序弥补了流行序列比对软件的不足，为PCR 设计引物、分析基因功能以及种质资源鉴定等方面提供新的工具。

关键词：VBA ；序列比对；保守序列；核酸数据库；大豆中图分类号：Q518．2文献标识码：A文章编号：1672－5565（2011）－04－299－04Searching conservative sequences in nuclear acid database by VBA programsZHAN Shao-Hua 1，YIN Yi-lin 1，CAI Yong-Ping 2，FAN Hong-Hong 2，LIN Yi 2*（1．Biological and Pharmacological Engineering Department ，West Anhui University ，Lu ’an ，Anhui 237012，China ；2．Life Science School ，Anhui Agricultural University ，Hefei Anhui 230036，China ）Abstract ：The four VBA （visual basic for application ）programs were written for searching conservative sequences in nuclear acid database．The programs included importing －DNA －sequence ，sorting －DNA －sequence ，DNA －random －sequence and finding －DNA －conservative －sequence．The DNA sequences saved as fasta format in text file could be imported into column A of Excel Sheet1by the program of importing －DNA －sequence ，at same time ，the Gi numbers were reserved and the redundant notes were deleted．Then ，the Gi numbers were sorted into column A and corresponding DNA integrate sequences were arranged into column B by the program of sorting －DNA －se-quence．DNA random sequences could be made by the program of DNA －random －sequence．The program of find-ing －DNA －conservative －sequence could help us searching conservative sequences in DNA databases by align-ment with the DNA random sequences．As an example of the programs application ，the conservative sequences of soybean genome survey sequences were searched．The programs were the supplementary tools of prevalent sequence alignment software ，could contribute to design PCR primers ，to analyze the genes function ，and to identify breeding resource．Key words ：Visual Basic for Application （VBA ）；Sequence alignment ；Conservative sequence ；Nuclear acid data-base ；Soybean序列比对是分子生物学中重要的分析方法，可用于探测新序列与已知序列的同源性，分析物种之间的亲缘关系［1］，可以在此基础上设计引物进行PCR 扩增、预测新序列高级结构、功能和基因电子克隆。

自噬双标腺病毒( mRFP-GFP-LC3 )使用指南背景：自噬是细胞内的一种“自食(Self-eating )”的现象，凋亡是“自杀( Self-killing )”的现象，二者共用相同的刺激因素和调节蛋白，但是诱发阈值和门槛不同，如何转换和协调目前还不清楚. 自噬是指膜(目前来源还有争议，大部分表现为双层膜，有时多层或单层)包裹部分胞质和细胞内需降解的细胞器、蛋白质等形成自噬体，最后与溶酶体融合形成自噬溶酶体，降解其所包裹的内容物，以实现细胞稳态和细胞器的更新。

目前文献对自噬过程进行观察和检测常用的策略和手段有：通过western blot检测LC3勺剪切；通过电镜观测自噬体的形成；在荧光显微镜下采用GFP(-RFP) -LC3等融合蛋白来示踪自噬体形成以及降解。

近几年对自噬流的研究日趋增多，针对于此我们汉恒生物科技(上海)有限公司自主研发了用于实时监测自噬( 流)的mRFP-GFP-LC腺病毒，mRFP用于标记及追踪LC3, GF啲减弱可指示溶酶体与自噬小体的融合形成自噬溶酶体，即由于GF荧光蛋白对酸性敏感，当自噬体与溶酶体融合后GFP 荧光发生淬灭, 此时只能检测到红色荧光。

这种串联的荧光蛋白表达载体系统直观清晰的指示了细胞自噬流的水平，是我们自噬研究尤其是自噬流研究不可或缺的利器。

mRFP-GFP-LC3 腺病毒的操作收到病毒后的处理（一）、腺病毒的储存1、腺病毒采用冰袋运输。

（1）、收到病毒液后如未融化请置于-80 C冰箱，下次使用时再进行分装；（2）、如客户收到时腺病毒已融化，请直接分装后置于-80 C冰箱保存；若短期内用于实验，可分装部分于4C保存（尽量一周内用完）。

2、尽量避免反复冻融，否则会降低病毒滴度（每次冻融会降低病毒滴度10%）。

建议不要在-20 C下长期保存。

如果病毒储存时间超过6个月，应该重新测定病毒滴度。

3、建议收到病毒产品后根据实验需求自行分装或购买经过分装的小包装病毒产品（购买时请提出）。

核农学报2024，38（2）：0226~0234Journal of Nuclear Agricultural Sciences苹果B型细胞分裂素响应因子MdARR11对干旱胁迫的抗性分析徐苏蕊1， 2赵文哲1， 2巩星遥1， 2李玲1， 2， *肖伟1， 2， *（1山东农业大学园艺科学与工程学院，山东泰安271018；2山东果蔬优质高效生产协同创新中心，山东泰安271018）摘要：B型反应调节因子（ARRs）作为细胞分裂素的正响应因子在植物生长发育中发挥重要作用。

为探究ARR11在应对苹果干旱胁迫过程中的功能，本研究以嘎拉3苹果（Malus domestica Borkh. cv. Gala 3）为试验材料，利用聚合酶链式反应（PCR）扩增技术，获得了B型细胞分裂素响应因子MdARR11。

该序列全长2 248 bp，编码613个氨基酸，包含type-B-REC结构域，C端含有一个MYB-like的DNA结合域。

组织特异性表达分析显示该基因在茎中表达量最高。

实时荧光定量PCR（qRT-PCR）分析结果表明，干旱胁迫抑制MdARR11的表达。

为进一步研究MdARR11在干旱胁迫中的功能，获得了过表达MdARR11苹果愈伤组织。

使用6%聚乙二醇6000（PEG6000）模拟干旱处理野生型及过表达愈伤组织，观察愈伤组织生长速率、大小并检测鲜重、相对电导率、丙二醛（MDA）、脯氨酸（Pro）、可溶性蛋白积累量以及超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性。

结果表明，过表达MdARR11提高了愈伤组织细胞膜的脂膜过氧化程度、降低了渗透调节物质的积累及抗氧化酶活性。

综上所述，MdARR11降低了苹果愈伤组织对干旱胁迫的耐受性。

本研究为进一步探索MdARR11基因的生物学功能及作用机理奠定了基础。

关键词：苹果；MdARR11； B型ARRs；干旱胁迫DOI：10.11869/j.issn.1000‑8551.2024.02.0226细胞分裂素（cytokinin，CTK）作为植物生长发育过程中不可或缺的经典激素之一，在植物中的信号传导由双组分系统（two-component system，TCS）介导［1］。

第46卷第6期2023年11月河北农业大学学报JOURNAL OF HEBEI AGRICULTURAL UNIVERSITYVol.46 No.6Nov.2023红树莓CPP转录因子家族的生物信息学及表达分析郑奕宸1，李 明1，李 闯1，吴菁菁1，李 寒2，顾玉红1（1.河北农业大学 生命科学学院，河北 保定 071001；2.河北农业大学 林学院，河北 保定 071001）摘要：为探索CPP转录因子家族在红树莓果实中的功能，本研究通过生物信息学技术与表达分析结合的方法，在‘海尔特兹’红树莓转录组数据库中检索到的CPP转录因子家族进行生物信息学分析、转录表达量分析和qRT-PCR表达分析。

结果表明：红树莓CPP基因家族包括4个成员，相对分子质量在47175.57～86763.46 kD之间，等电点在5.83～9.20之间，为不稳定、亲水性的蛋白质，无信号肽，定位于细胞核，无跨膜结构，含有2个CXC保守结构域，结构主要由无规则卷曲构成，含有8～12个外显子，7～8个保守基序，与拟南芥的CPP基因亲缘关系较近，含有生长素、脱落酸、赤霉素、水杨酸、防御和应激、干旱诱导等顺式作用元件，转录表达和qRT-PCR表达均分析发现RuCPP-1和RuCPP-4基因在青果时期的表达量最高，RuCPP-2和RuCPP-3基因分别在红果和深红果时期的表达量最高，推测RuCPP基因家族可能参与调控生长素、脱落酸、赤霉素、水杨酸等反应。

关 键 词：红树莓；CPP转录因子家族；生物信息学分析；表达分析中图分类号：Q781；S663.2开放科学（资源服务）标识码（OSID）：文献标志码：ABioinformatics and expression analysis of CPP transcription factorfamily of red raspberryZHENG Yichen1, LI Ming1, LI Chuang1, WU Jingjing1, LI Han2, GU Yuhong1(1. College of Life Science, Hebei Agricultural University , Baoding 071001, China; 2. College of Foresty, HebeiAgricultural University , Baoding 071001, China)Abstract: In order to explore the function of CPP transcription factor family in red raspberry fruits, this studyconducted bioinformatics analysis, transcriptional expression analysis and qRT-PCR expression analysis of CPPtranscription factor family retrieved from the ‘Heritage’ red raspberry transcriptome database. The results showedthat the red raspberry CPP gene family consisted of 4 members, whose relative molecular weight ranged from47 175.57 to 86 763.46 kD and isoelectric point ranged from 5.83 to 9.20. They were unstable, hydrophilic proteinswithout signal peptides, and localized on the nucleus without transmembrane structure. The CPP proteins contained2 CXC conserved domains and the structure were mainly composed of random crimp. The CPP genes contained8～12 exons and 7～8 conserved motifs. They were closely related to the CPP gene of Arabidopsis thaliana.Cis-acting elements were identified including auxin, abscisic acid, gibberellin, salicylic acid, drought induction,defense and stress responsive elements. Expression analysis showed that the expression levels of RuCPP-1 andRuCPP-4 were the higher in the green fruit stage, and the expression levels of RuCPP-2 and RuCPP -3 were the收稿日期：2023-07-26基金项目：河北省重点研发计划项目（20326338D）.第一作者：郑奕宸（1999-），女，河北邢台人，硕士研究生，从事植物发育生物学研究.E-mail:**********************通信作者：顾玉红（1977—），女，河北昌黎人，博士，教授，从事植物发育生物学研究.E-mail:******************本刊网址：文章编号：1000-1573(2023)06-0058-08DOI：10.13320/ki.jauh.2023.009359第6期higher in the red and deep red fruit stages. It was speculated that RuCPP gene family might be involved in the response of auxin, abscisic acid, gibberellin and salicylic acid.Keywords: red raspberry; CPP transcription factor family; bioinformatics analysis; expression analysis红树莓（Rubus idaeus L.）又名覆盆子、悬钩子、托盘等，属于蔷薇科悬钩子属，在寒带和温带各地均有分布［1］。

㊀山东农业科学㊀２０２３ꎬ５５(３):９~１４ＳｈａｎｄｏｎｇＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓ㊀ＤＯＩ:１０.１４０８３/ｊ.ｉｓｓｎ.１００１－４９４２.２０２３.０３.００２收稿日期:２０２２－０６－２８基金项目:山东省农业良种工程项目(２０１９ＬＺＧＣ０１７０２)ꎻ山东省自然科学基金青年项目(ＺＲ２０２０ＱＣ１１４)ꎻ国家自然科学基金青年项目(３２００１５４２)ꎻ山东省农业良种工程项目(２０２１ＬＺＧＣ０１３)ꎻ小麦玉米国家工程实验室开放课题(２０１８ＬＹＺＷＳ０６)作者简介:李永波(１９８６ )ꎬ男ꎬ博士ꎬ助理研究员ꎬ主要从事小麦新品种培育研究ꎮＥ－ｍａｉｌ:ｌｙｂ９２０３２７＠ｓｉｎａ.ｃｏｍ通信作者:樊庆琦(１９８０ )ꎬ男ꎬ博士ꎬ副研究员ꎬ主要从事小麦新品种培育研究ꎮＥ－ｍａｉｌ:ｆａｎｑｉｎｇｑｉ＠１６３.ｃｏｍ楚秀生(１９６３ )ꎬ男ꎬ博士ꎬ研究员ꎬ主要从事小麦新品种培育研究ꎮＥ－ｍａｉｌ:ｘｓｃｈｕ２００７＠ｓｉｎａ.ｃｏｍ小麦ＤＲＥＢ４蛋白的原核表达及多克隆抗体制备李永波１ꎬ鲁琳１ꎬ方会见２ꎬ崔德周１ꎬ孟福燕３ꎬ黄琛１ꎬ隋新霞１ꎬ樊庆琦１ꎬ楚秀生１ꎬ４(１.山东省农业科学院作物研究所/黄淮北部小麦生物学与遗传育种重点实验室/山东省小麦技术创新中心/济南市小麦遗传改良重点实验室ꎬ山东济南㊀２５０１００ꎻ２.山东鲁研良种有限公司ꎬ山东济南㊀２５０１００ꎻ３.郓城县种子公司ꎬ山东郓城㊀２７４７００ꎻ４.烟台大学生命科学学院ꎬ山东烟台㊀２６４０００)㊀㊀摘要:ＤＲＥＢ(ｄｅｈｙｄｒａｔｉｏｎｒｅｓｐｏｎｓｉｖｅｅｌｅｍｅｎｔｂｉｎｄｉｎｇ)转录因子在小麦非生物胁迫中起着非常重要的作用ꎬ但由于目前缺乏可识别小麦内源性ＤＲＥＢ蛋白的抗体ꎬ导致其在蛋白水平上的研究进展非常缓慢ꎮ本研究通过分析ＤＲＥＢ４Ａ㊁４Ｂ和４Ｃ三种蛋白序列ꎬ将ＤＲＥＢ４Ａ在大肠杆菌中进行表达ꎬ并利用纯化后的蛋白作为抗原免疫兔子ꎬ在国内外首次获得小麦ＤＲＥＢ４的多克隆抗体ꎮＷｅｓｔｅｒｎｂｌｏｔ结果证明ꎬ该抗体可特异性识别小麦内源性ＤＲＥＢ４蛋白ꎮ该抗体介导的免疫组织化学结果显示ꎬＤＲＥＢ４蛋白定位于细胞核内ꎮ本研究为深入研究植物ＤＲＥＢ信号通路提供了有力的检测工具ꎮ关键词:小麦ꎻ非生物胁迫ꎻＤＲＥＢ４转录因子ꎻ多克隆抗体中图分类号:Ｓ５１２.１:Ｑ７８６㊀㊀文献标识号:Ａ㊀㊀文章编号:１００１－４９４２(２０２３)０３－０００９－０６ＰｒｏｋａｒｙｏｔｉｃＥｘｐｒｅｓｓｉｏｎａｎｄＰｏｌｙｃｌｏｎａｌＡｎｔｉｂｏｄｙＰｒｅｐａｒａｔｉｏｎｏｆＷｈｅａｔＤＲＥＢ４ＰｒｏｔｅｉｎＬｉＹｏｎｇｂｏ１ꎬＬｕＬｉｎ１ꎬＦａｎｇＨｕｉｊｉａｎ２ꎬＣｕｉＤｅｚｈｏｕ１ꎬＭｅｎｇＦｕｙａｎ３ꎬＨｕａｎｇＣｈｅｎ１ꎬＳｕｉＸｉｎｘｉａ１ꎬＦａｎＱｉｎｇｑｉ１ꎬＣｈｕＸｉｕｓｈｅｎｇ１ꎬ４(１.ＣｒｏｐＲｅｓｅａｒｃｈＩｎｓｔｉｔｕｔｅꎬＳｈａｎｄｏｎｇＡｃａｄｅｍｙｏｆＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓ/ＫｅｙＬａｂｏｒａｔｏｒｙｏｆＷｈｅａｔＢｉｏｌｏｇｙａｎｄＧｅｎｅｔｉｃｓａｎｄＢｒｅｅｄｉｎｇｉｎＮｏｒｔｈｅｒｎＨｕａｎｇ￣ＨｕａｉＲｉｖｅｒＰｌａｉｎꎬＭｉｎｉｓｔｒｙｏｆＡｇｒｉｃｕｌｔｕｒｅａｎｄＲｕｒａｌＡｆｆａｉｒｓ/ＳｈａｎｄｏｎｇＴｅｃｈｎｏｌｏｇｙＩｎｎｏｖａｔｉｏｎＣｅｎｔｅｒｏｆＷｈｅａｔ/ＪｉｎａｎＫｅｙＬａｂｏｒａｔｏｒｙｏｆＷｈｅａｔＧｅｎｅｔｉｃＩｍｐｒｏｖｅｍｅｎｔꎬＪｉｎａｎ２５０１００ꎬＣｈｉｎａꎻ２.ＳｈａｎｄｏｎｇＬｕｙａｎＳｅｅｄＣｏ.ꎬＬｔｄ.ꎬＪｉｎａｎ２５０１００ꎬＣｈｉｎａꎻ３.ＹｕｎｃｈｅｎｇＣｏｕｎｔｒｙＳｅｅｄＣｏｍｐａｎｙꎬＹｕｎｃｈｅｎｇ２７４７００ꎬＣｈｉｎａꎻ４.ＣｏｌｌｅｇｅｏｆＬｉｆｅＳｃｉｅｎｃｅｓꎬＹａｎｔａｉＵｎｉｖｅｒｓｉｔｙꎬＹａｎｔａｉ２６４０００ꎬＣｈｉｎａ)Ａｂｓｔｒａｃｔ㊀ＤＲＥＢ(ｄｅｈｙｄｒａｔｉｏｎ￣ｒｅｓｐｏｎｓｉｖｅｅｌｅｍｅｎｔ￣ｂｉｎｄｉｎｇ)ｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｐｌａｙｓａｖｅｒｙｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｗｈｅａｔａｂｉｏｔｉｃｓｔｒｅｓｓ.ＨｏｗｅｖｅｒꎬｄｕｅｔｏｔｈｅｌａｃｋｏｆａｎｔｉｂｏｄｉｅｓｔｈａｔｃａｎｒｅｃｏｇｎｉｚｅｗｈｅａｔｅｎｄｏｇｅｎｏｕｓＤＲＥＢｐｒｏｔｅｉｎꎬｉｔｓｒｅｓｅａｒｃｈｐｒｏｇｒｅｓｓａｔｐｒｏｔｅｉｎｌｅｖｅｌｉｓｖｅｒｙｓｌｏｗ.ＩｎｔｈｉｓｓｔｕｄｙꎬｂｙａｎａｌｙｚｉｎｇｔｈｒｅｅｐｒｏｔｅｉｎｓｅｑｕｅｎｃｅｓｏｆＤＲＥＢ４Ａꎬ４Ｂａｎｄ４ＣꎬｔｈｅＤＲＥＢ４ＡｗａｓｓｅｌｅｃｔｅｄｔｏｅｘｐｒｅｓｓｉｎＥｓｃｈｅｒｉｃｈｉａｃｏｌｉꎬａｎｄｔｈｅｐｕｒｉｆｉｅｄｐｒｏｔｅｉｎｗａｓｕｓｅｄａｓａｎａｎｔｉｇｅｎｔｏｉｍｍｕｎｉｚｅｒａｂｂｉｔｓꎬｔｈｅｎｔｈｅｐｏｌｙｃｌｏｎａｌａｎｔｉｂｏｄｙｏｆｗｈｅａｔＤＲＥＢ４ｗａｓｏｂｔａｉｎｅｄｆｏｒｔｈｅｆｉｒｓｔｔｉｍｅａｔｈｏｍｅａｎｄａｂｒｏａｄ.ＴｈｅＷｅｓｔｅｒｎｂｌｏｔｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔｔｈｅａｎｔｉｂｏｄｙｃｏｕｌｄｓｐｅｃｉｆｉｃａｌｌｙｒｅｃｏｇｎｉｚｅｗｈｅａｔｅｎｄｏｇｅｎｏｕｓＤＲＥＢ４ｐｒｏｔｅｉｎ.Ｔｈｅａｎｔｉｂｏｄｙ￣ｍｅｄｉａｔｅｄｉｍｍｕｎｏｈｉｓｔｏｃｈｅｍｉｃａｌｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔＤＲＥＢ４ｐｒｏｔｅｉｎｗａｓｌｏｃａｌｉｚｅｄｉｎｔｈｅｎｕｃｌｅｕｓ.Ｔｈｉｓｓｔｕｄｙｃｏｕｌｄｐｒｏｖｉｄｅａｐｏｗｅｒｆｕｌｄｅｔｅｃｔｉｏｎｔｏｏｌｆｏｒｉｎ￣ｄｅｐｔｈｒｅｓｅａｒｃｈｏｆｐｌａｎｔＤＲＥＢｓｉｇｎａｌｉｎｇｐａｔｈｗａｙ.Ｋｅｙｗｏｒｄｓ㊀ＷｈｅａｔꎻＡｂｉｏｔｉｃｓｔｒｅｓｓꎻＤＲＥＢ４ｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒꎻＰｏｌｙｃｌｏｎａｌａｎｔｉｂｏｄｙ㊀㊀干旱㊁盐㊁高温㊁冷等各种非生物胁迫会严重影响小麦产量ꎮＤＲＥＢ蛋白含有一个保守的ＡＰ２结构域ꎬ可以和顺式作用元件ＤＲＥ核心序列(Ａ/ＧＣＣＧＡＣ)发生特异性结合ꎬ通过在转录水平上调控下游基因的表达[１]ꎬ进而应对各种非生物胁迫ꎮ到目前为止ꎬＤＲＥＢ转录因子在拟南芥[２]㊁大豆[３]㊁水稻[４]㊁玉米[５]㊁大麦[６]和小麦[７]等多种植物中被鉴定出来ꎮＤＲＥＢ分为六大类(ＤＲＥＢ１~６)[８]ꎬ其中ꎬＤＲＥＢ１在拟南芥㊁水稻㊁玉米中主要应答冷胁迫[４]ꎬＤＲＥＢ２主要应答干旱㊁盐胁迫[９]ꎬＤＲＥＢ３参与ＡＢＡ和糖信号途径[１０]ꎬＤＲＥＢ４应答干旱㊁冷胁迫及在乙烯与茉莉酸途径中起作用[１１]ꎬＤＲＥＢ５参与应答干旱㊁冷胁迫[１２]ꎬＤＲＥＢ６应答干旱㊁盐胁迫[１３]ꎮ大量研究已验证了ＤＲＥＢ在植物应对非生物胁迫中的功能ꎮ如在小麦中过表达拟南芥ＤＲＥＢ１Ａ㊁大豆ＧｍＤＲＥＢ１或棉花ＧｈＤＲＥＢ基因ꎬ可通过提高根系活力㊁光合作用及渗透调节能力提高小麦的抗旱性[１４－１６]ꎻ在拟南芥中过表达大豆ＧｍＤＲＥＢ２㊁ＧｍＤＲＥＢ３或小麦ＴａＤＲＥＢ３基因ꎬ可提高拟南芥抗旱㊁耐盐㊁耐高温及抗冻性[１ꎬ１７ꎬ１８]ꎻ过表达ＧｍＤＲＥＢ６基因ꎬ可增强大豆的耐盐能力[１９]ꎮ然而ꎬ目前几乎所有关于ＤＲＥＢ的研究是集中在转录水平上的调控ꎬ缺乏蛋白质水平上的调控研究ꎬ且ＤＲＥＢ蛋白发挥生物学功能是否通过磷酸化㊁乙酰化等蛋白水平上的调控尚未可知ꎬ因此ꎬ研究识别内源性ＤＲＥＢ蛋白的特异性多克隆抗体ꎬ对于ＤＲＥＢ在蛋白水平的调控研究具有非常重要的意义ꎮ本研究通过对小麦中已有的ＤＲＥＢ４Ａ㊁４Ｂ和４Ｃ进行序列分析ꎬ选取ＤＲＥＢ４Ａ进行原核表达㊁纯化ꎬ并以其作为抗原ꎬ首次制备出可识别小麦内源ＤＲＥＢ４蛋白的多克隆抗体ꎬ以期为进一步研究植物ＤＲＥＢ４在蛋白水平上的调控机理提供方法学基础ꎮ１㊀材料与方法１.１㊀试验材料供试小麦品种为济麦３７９ꎬ由山东省审定(鲁审麦２０２１００１７)ꎮ取其幼苗期根㊁叶为材料进行试验ꎮ１.２㊀ＤＲＥＢ４序列分析与合成本研究通过ＤＮＡＭＡＮ８软件对ＮＣＢＩ中提交的ＤＲＥＢ４Ａ(ＡＹ７８１３５４.１)㊁４Ｂ(ＡＹ７８１３５５.１)和４Ｃ(ＡＹ７８１３５６.１)序列进行分析ꎻＤＲＥＢ４Ａ序列的合成由北京擎科生物科技有限公司进行ꎮ１.３㊀ＤＲＥＢ４Ａ载体构建、原核表达及纯化将上述合成的ＤＲＥＢ４Ａ序列与大肠杆菌表达载体ＰＥＴ３０ａ通过同源重组的方法(ｐＥＡＳＹ®－ＢａｓｉｃＳｅａｍｌｅｓｓＣｌｏｎｉｎｇａｎｄＡｓｓｅｍｂｌｙＫｉｔꎬＣＵ２０１－０２ꎬ北京全式金生物技术股份有限公司)连接ꎬ将连接产物转入ＤＨ５α(北京擎科生物科技有限公司)感受态细胞中ꎬ冰上放置１５ｍｉｎꎬ４２ħ水浴热激９０ｓꎬ再冰上放置２ｍｉｎꎬ加入１ｍＬ无任何抗生素的ＬＢ液体培养基ꎬ３７ħ㊁２１０ｒ/ｍｉｎ水平摇１ｈꎬ然后取１００μＬ菌液ꎬ涂于含有卡那霉素的ＬＢ固体培养基上ꎬ３７ħ过夜培养ꎮ挑取１０个单克隆进行ＰＣＲ检测ꎬ选取２个阳性信号最强的单克隆由北京擎科生物科技有限公司进行测序ꎬ对测序正确的单克隆进行摇菌㊁质粒提取(质粒小提试剂盒ꎬＤＰ１０３ꎬ北京天根生化科技有限公司)ꎮ将提取好的质粒转入ＢＬ２１(ＤＥ３)感受态细胞(ＣＤ７０１－０２ꎬ北京全式金生物技术股份有限公司)中ꎬ剩余步骤同ＤＨ５α感受态细胞转化ꎮ挑单克隆ꎬ置于５ｍＬＬＢ液体培养基中ꎬ３７ħ过夜培养ꎬ然后吸取１ｍＬ菌液ꎬ加入到３００ｍＬＬＢ液体培养基中进行扩大培养ꎬ待菌液ＯＤ值为０.６~０.８时ꎬ加入终浓度为５０ｍｍｏｌ/Ｌ的ＩＰＴＧ(Ｇ５０４２－１Ｇꎬ武汉塞维尔生物科技有限公司)进行诱导表达ꎬ２８ħ过夜培养ꎻ菌液于６０００ｒ/ｍｉｎ离心１０ｍｉｎꎬ收集菌体沉淀ꎬ用１ˑＰＢＳ(ｐｈｏｓｐｈａｔｅｂｕｆｆｅｒｓａｌｉｎｅ)清洗沉淀１次ꎬ然后加入４０ｍＬ１ˑＰＢＳ重悬ꎬ超声破碎(开３ｓꎬ关３ｓꎻ共计３０ｍｉｎ)ꎬ６０００ｒ/ｍｉｎ离心１０ｍｉｎꎬ分别将沉淀㊁上清液进行ＳＤＳ电泳检测ꎮ利用Ｈｉｓ标签蛋白纯化试剂盒(Ｐ２２２６ꎬ上海碧云天生物技术有限公司)对上清液进行纯化ꎬ然后置于透析袋中４ħ过夜透析ꎬ将透析后的蛋白置于０１山东农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀第５５卷㊀－２０ħ保存备用ꎮ１.４㊀小麦ＤＲＥＢ４多克隆抗体制备选取实验级日本大耳白兔和新西兰大白兔各１只ꎬ饲养体重至１~２ｋｇ时ꎬ用注射器将充分混匀的１ｍＬ完全弗氏佐剂(液体石蜡ʒ羊毛脂＝２ʒ１)和０.３ｍｇＤＲＥＢ４Ａ融合蛋白对每只兔子进行皮下注射第１针ꎬ标记为第１天ꎻ第１２天ꎬ将充分混匀的１ｍＬ不完全弗氏佐剂(完全弗氏佐剂＋终浓度２０ｍｇ/ｍＬ的卡介苗)和０.１５ｍｇ融合蛋白对每只兔子进行皮下注射第２针ꎻ第２６天ꎬ将充分混匀的１ｍＬ不完全弗氏佐剂和０.１５ｍｇ融合蛋白对每只兔子进行肌肉注射第３针ꎻ第４０天ꎬ将充分混匀的１ｍＬ不完全弗氏佐剂和０.１５ｍｇ融合蛋白对每只兔子进行肌肉注射第４针ꎻ第５３天ꎬ取兔子血清进行Ｗｅｓｔｅｒｎｂｌｏｔ验证ꎮ１.５㊀Ｗｅｓｔｅｒｎｂｌｏｔ分析利用植物组织蛋白裂解液提取小麦幼苗期根㊁叶部的总蛋白(植物蛋白提取试剂盒ꎬＣＷ０８８５ꎬ康为世纪生物技术有限公司)ꎬ配制１５％的聚丙烯酰胺凝胶进行电泳ꎻ通过湿法转膜ꎬ将凝胶中的蛋白转移到硝酸纤维素薄膜上ꎬ然后将膜放入含有２％脱脂奶粉的ＴＢＳ(２５ｍｍｏｌ/ＬＴｒｉｓ－ＨＣｌꎬ１３７ｍｍｏｌ/ＬＮａＣｌ)中ꎬ封闭１ｈꎻ加入ＤＲＥＢ４多克隆抗体(１ʒ１０００稀释于２％脱脂奶粉中)ꎬ４ħ过夜ꎻ用ＴＢＳＴ(ＴＢＳ＋２０％吐温－２０)洗涤３次后ꎬ向封闭液中加入碱性磷酸酶(ａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅꎬＡＰ)标记的二抗ꎬ缓慢摇动２４ｈꎬ然后ＴＢＳＴ洗涤３次ꎬ每次１０ｍｉｎꎻ最后用发色液(ＴＢＳ１０ｍＬꎬ５％ＮＢＴ４５μＬꎬ５％ＢＣＩＰ３５μＬ)进行发色ꎮ１.６㊀免疫组织化学法进行亚细胞定位将小麦叶片下表皮撕下ꎬ置于４％多聚甲醛中ꎬ室温放置２４ｈꎬ弃掉多聚甲醛ꎬ用１ˑＰＢＳ清洗３次ꎬ加入２％脱脂奶粉于３７ħ封闭３０ｍｉｎꎬ然后在４ħ下加入ＤＲＥＢ４多克隆抗体(１ʒ２００稀释于２％脱脂奶粉中)过夜ꎻ用ＰＢＳ洗涤３次后ꎬ加入１μＬ二抗(山羊抗兔－ＡｌｅｘａＦｌｕｏｒ５５５抗体)和１０ｍＬＢＳＡꎬ３７ħ继续孵育１ｈꎬ然后用ＴＢＳ清洗３次ꎬ在室温下用４ᶄꎬ６－二脒基－２－苯基吲哚(ＤＡ￣ＰＩꎬＡｎａＳｐｅｃＩｎｃ.ꎬＳａｎＪｏｓｅꎬＣＡꎬＵＳＡ)染色１０ｍｉｎꎬ然后用ＴＢＳ清洗３次ꎬ置于荧光显微镜(ＨＴ７７００ꎬＨｉｔａｃｈｉꎬＴｏｋｙｏꎬＪａｐａｎ)下观察并拍照ꎮ２㊀结果与分析２.１㊀小麦ＤＲＥＢ４序列分析在普通小麦中ꎬＤＲＥＢ４存在ＤＲＥＢ４Ａ㊁４Ｂ㊁４Ｃ三种转录本ꎬ其中ꎬＤＲＥＢ４Ａ编码３９４个氨基酸ꎬ分子量为４２.８ｋＤａꎻＤＲＥＢ４Ｂ编码３４６个氨基酸ꎬ分子量为３７.７ｋＤａꎻＤＲＥＢ４Ｃ编码６８个氨基酸ꎬ分子量为７.１ｋＤａ(图１)ꎮ三个蛋白氨基酸序列的保守性为６１.２８％ꎬ第１~２５位的氨基酸完全一致ꎬ其中ꎬＤＲＥＢ４Ｂ除第２６~７３位氨基酸缺失外ꎬ其它位置的氨基酸与ＤＲＥＢ４Ａ完全一致ꎮＤＲＥＢ４Ａ㊁４Ｂ和４Ｃ存在序列间的差异ꎬ可能是应对不同非生物胁迫产生的可变剪切所致ꎮ图中深蓝色区域为保守区域ꎮ图１㊀普通小麦ＤＲＥＢ４Ａ、４Ｂ和４Ｃ的氨基酸序列分析２.２㊀小麦ＤＲＥＢ４Ａ的原核表达鉴于ＤＲＥＢ４Ａ的氨基酸序列最长ꎬ选其进行后续分析ꎮ首先ꎬ将人工合成的ＤＲＥＢ４Ａ序列与表达载体ＰＥＴ３０ａ连接后ꎬ在大肠杆菌中进行表达ꎬ上清液中的蛋白纯化后进行ＳＤＳ－ＰＡＧＥ检测ꎮ结果显示ꎬ在大约５０ｋＤａ处出现清晰的蛋白条带(图２)ꎬ与预期的蛋白分子量相符ꎬ表明ＤＲＥＢ４Ａ成功表达ꎮ１１㊀第３期㊀㊀㊀㊀㊀㊀㊀李永波ꎬ等:小麦ＤＲＥＢ４蛋白的原核表达及多克隆抗体制备图２㊀小麦ＤＲＥＢ４Ａ的原核表达及纯化２.３㊀ＤＲＥＢ４多克隆抗体的制备本研究以上述获得的纯化ＤＲＥＢ４Ａ融合蛋白为抗原免疫兔子ꎬ从兔血清中获取了ＤＲＥＢ４多克隆抗体ꎮＷｅｓｔｅｒｎｂｌｏｔ结果显示ꎬ该抗体在目的蛋白位置清楚地识别到ＤＲＥＢ４Ａ蛋白(图３)ꎮ图３㊀ＤＲＥＢ４多克隆抗体对ＤＲＥＢ４Ａ融合蛋白的识别２.４㊀ＤＲＥＢ４多克隆抗体对小麦内源性ＤＲＥＢ４蛋白的识别及特异性检测为了进一步验证该抗体能否识别小麦内源性ＤＲＥＢ４蛋白ꎬ分别提取小麦苗期根㊁叶总蛋白进行免疫识别ꎮＷｅｓｔｅｒｎｂｌｏｔ结果显示ꎬ仅在３７ｋＤａ处检测到清晰的蛋白条带ꎬ这与预测的ＤＲＥＢ４Ｂ蛋白分子量一致(图４)ꎮ表明该抗体可以识别小麦内源性ＤＲＥＢ４Ｂ蛋白ꎬ而且特异性好ꎬ可以用于后续植物ＤＲＥＢ分子机理的相关研究ꎮ图４㊀小麦内源性ＤＲＥＢ４蛋白检测２.５㊀ＤＲＥＢ４亚细胞定位分析ＤＲＥＢ４定位于细胞核中(图５)ꎬ与前人报道的ＤＲＥＢ转录因子核定位的结果一致[２０]ꎬ进一步证实了该抗体特异性较好ꎬ可用于开展免疫组织或细胞化学研究的可行性ꎮ对照为前血清ꎮ图５㊀利用免疫组织化学法进行的㊀㊀㊀ＤＲＥＢ４亚细胞定位分析结果３㊀讨论与结论ＤＲＥＢ是一类抗非生物胁迫的转录因子ꎬ目前主要用于抗逆转基因植物的培育及相关分子机理的解析[２１]ꎮ小麦ＤＲＥＢ４蛋白是一种对动物和人类无危害的蛋白ꎬ将其用于粮食作物抗逆性转基因改良有着广阔的市场前景[２２]ꎮＤＲＥＢ４在小麦中存在三种转录形式(ＤＲＥＢ４Ａ㊁４Ｂ和４Ｃ)[２３]ꎬ其中ꎬＤＲＥＢ４Ａ编码的多肽链最长ꎬ涵盖的蛋白信２１山东农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀第５５卷㊀息最丰富ꎬ推测由此蛋白作为抗原产生的抗体可识别ＤＲＥＢ４的所有三种形式ꎬ因此本研究利用ＤＲＥＢ４Ａ蛋白作为抗原ꎬ进行了ＤＲＥＢ４多克隆抗体的制备ꎮ经过抗原上清蛋白的纯化㊁免疫注射ꎬ最终研制出能识别小麦内源性ＤＲＥＢ４蛋白的多克隆抗体ꎮ尽管从植物中已克隆出多种类型的ＤＲＥＢ基因ꎬ但由于其抗体类型匮乏以及识别内源性蛋白抗体的空白ꎬ导致有关ＤＲＥＢ在蛋白水平上的调控机理研究进展相对缓慢ꎮ目前ꎬ只有拟南芥ＤＲＥＢ１Ａ的抗体制备成功ꎬ且仅对大肠杆菌中表达的拟南芥ＤＲＥＢ１Ａ融合蛋白进行了检测[２４]ꎮ本研究首次开发了特异性识别小麦内源ＤＲＥＢ４蛋白的多克隆抗体ꎬ既丰富了植物ＤＲＥＢ的抗体类型ꎬ也为进一步推动ＤＲＥＢ在蛋白水平上的研究提供了方法学基础ꎮ利用本研究制备的ＤＲＥＢ４多克隆抗体检测小麦苗期根㊁叶内源性ＤＲＥＢ４蛋白时ꎬ仅识别到了ＤＲＥＢ４Ｂ蛋白条带ꎬ与预测的该抗体能识别小麦中ＤＲＥＢ４三种蛋白形式的结果不一致ꎬ这可能是因为ＤＲＥＢ４具有组织器官以及不同发育阶段表达特异性ꎬ在小麦苗期根㊁叶中主要以ＤＲＥＢ４Ｂ的形式表达ꎬ而在花㊁籽粒等其它组织器官以及不同发育阶段中则以其它形式表达ꎻ另外ꎬＤＲＥＢ４在不同小麦品种中的表达形式也可能存在一定的差异ꎬ本研究所用小麦品种济麦３７９为抗旱节水型品种ꎬ在其苗期根㊁叶中主要以ＤＲＥＢ４Ｂ的形式表达ꎬ但在其它类型的小麦品种中以哪种形式表达还有待进一步研究ꎮ传统ＤＲＥＢ基因亚细胞定位是采用构建ＤＲＥＢ－ＧＦＰ过表达载体转入组织或细胞中的方法进行定位[２０]ꎬ而本研究是利用该抗体对内源ＤＲＥＢ４进行免疫定位ꎬ与传统方法相比可避免因过表达造成目的蛋白移位的现象ꎮ综上所述ꎬ本研究通过对ＤＲＥＢ４Ａ进行大肠杆菌表达㊁纯化ꎬ并以此作为抗原成功制备出可识别小麦内源性ＤＲＥＢ４蛋白的高度特异性多克隆抗体ꎬ可为深入研究植物ＤＲＥＢ４在蛋白水平上参与非生物胁迫的调控机理奠定方法学基础ꎮ参㊀考㊀文㊀献:[１]㊀ＮｉｕＸꎬＬｕｏＴꎬＺｈａｏＨꎬｅｔａｌ.ＩｄｅｎｔｉｆｉｃａｔｉｏｎｏｆｗｈｅａｔＤＲＥＢｇｅｎｅｓａｎｄｆｕｎｃｔｉｏｎａｌｃｈａｒａｃｔｅｒｉｚａｔｉｏｎｏｆＴａＤＲＥＢ３ｉｎｒｅｓｐｏｎｓｅｔｏａｂｉｏｔｉｃｓｔｒｅｓｓｅｓ[Ｊ].Ｇｅｎｅꎬ２０２０ꎬ７４０:１４４５１４. [２]㊀ＳｔｏｃｋｉｎｇｅｒＥＪꎬＧｉｌｍｏｕｒＳＪꎬＴｈｏｍａｓｈｏｗＭＦ.ＡｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａＣＢＦ１ｅｎｃｏｄｅｓａｎＡＰ２ｄｏｍａｉｎ￣ｃｏｎｔａｉｎｉｎｇｔｒａｎｓｃｒｉｐ￣ｔｉｏｎａｌａｃｔｉｖａｔｏｒｔｈａｔｂｉｎｄｓｔｏｔｈｅＣ￣ｒｅｐｅａｔ/ＤＲＥꎬａｃｉｓ￣ａｃｔｉｎｇＤＮＡｒｅｇｕｌａｔｏｒｙｅｌｅｍｅｎｔｔｈａｔｓｔｉｍｕｌａｔｅｓｔｒａｎｓｃｒｉｐｔｉｏｎｉｎｒｅ￣ｓｐｏｎｓｅｔｏｌｏｗｔｅｍｐｅｒａｔｕｒｅａｎｄｗａｔｅｒｄｅｆｉｃｉｔ[Ｊ].Ｐｒｏｃ.Ｎａｔｌ.Ａｃａｄ.Ｓｃｉ.ＵＳＡꎬ１９９７ꎬ９４(３):１０３５－１０４０. [３]㊀ＭｉｚｏｉＪꎬＯｈｏｒｉＴꎬＭｏｒｉｗａｋｉＴꎬｅｔａｌ.ＧｍＤＲＥＢ２Ａꎻ２ꎬａｃａｎｏｎｉｃａｌＤＥＨＹＤＲＡＴＩＯＮ￣ＲＥＳＰＯＮＳＩＶＥＥＬＥＭＥＮＴ￣ＢＩＮＤＩＮＧＰＲＯＴＥＩＮ２￣ｔｙｐｅｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｉｎｓｏｙｂｅａｎꎬｉｓｐｏｓｔｔｒａｎｓｌａｔｉｏｎａｌｌｙｒｅｇｕ￣ｌａｔｅｄａｎｄｍｅｄｉａｔｅｓｄｅｈｙｄｒａｔｉｏｎ￣ｒｅｓｐｏｎｓｉｖｅｅｌｅｍｅｎｔ￣ｄｅｐｅｎｄｅｎｔｇｅｎｅｅｘｐｒｅｓｓｉｏｎ[Ｊ].ＰｌａｎｔＰｈｙｓｉｏｌ.ꎬ２０１３ꎬ１６１(１):３４６－３６１.[４]㊀ＤｕｂｏｕｚｅｔＪＧꎬＳａｋｕｍａＹꎬＩｔｏＹꎬｅｔａｌ.ＯｓＤＲＥＢｇｅｎｅｓｉｎｒｉｃｅꎬＯｒｙｚａｓａｔｉｖａＬ.ꎬｅｎｃｏｄｅｔｒａｎｓｃｒｉｐｔｉｏｎａｃｔｉｖａｔｏｒｓｔｈａｔｆｕｎｃｔｉｏｎｉｎｄｒｏｕｇｈｔ￣ꎬｈｉｇｈ￣ｓａｌｔ￣ａｎｄｃｏｌｄ￣ｒｅｓｐｏｎｓｉｖｅｇｅｎｅｅｘｐｒｅｓｓｉｏｎ[Ｊ].ＰｌａｎｔＪ.ꎬ２００３ꎬ３３(４):７５１－７６３.[５]㊀ＱｉｎＦꎬＫａｋｉｍｏｔｏＭꎬＳａｋｕｍａＹꎬｅｔａｌ.Ｒｅｇｕｌａｔｉｏｎａｎｄｆｕｎｃｔｉｏｎ￣ａｌａｎａｌｙｓｉｓｏｆＺｍＤＲＥＢ２ＡｉｎｒｅｓｐｏｎｓｅｔｏｄｒｏｕｇｈｔａｎｄｈｅａｔｓｔｒｅｓｓｅｓｉｎＺｅａｍａｙｓＬ.[Ｊ].ＰｌａｎｔＪ.ꎬ２００７ꎬ５０(１):５４－６９. [６]㊀ＸｕｅＧＰ.ＡｎＡＰ２ｄｏｍａｉｎｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒＨｖＣＢＦ１ａｃｔｉ￣ｖａｔｅｓｅｘｐｒｅｓｓｉｏｎｏｆｃｏｌｄ￣ｒｅｓｐｏｎｓｉｖｅｇｅｎｅｓｉｎｂａｒｌｅｙｔｈｒｏｕｇｈｉｎ￣ｔｅｒａｃｔｉｏｎｗｉｔｈａ(Ｇ/ａ)(Ｃ/ｔ)ＣＧＡＣｍｏｔｉｆ[Ｊ].Ｂｉｏｃｈｉｍ.Ｂｉｏ￣ｐｈｙｓ.Ａｃｔａꎬ２００２ꎬ１５７７(１):６３－７２.[７]㊀ＳｈｅｎＹＧꎬＺｈａｎｇＷＫꎬＨｅＳＪꎬｅｔａｌ.ＡｎＥＲＥＢＰ/ＡＰ２￣ｔｙｐｅｐｒｏｔｅｉｎｉｎＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍｗａｓａＤＲＥ￣ｂｉｎｄｉｎｇｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｉｎｄｕｃｅｄｂｙｃｏｌｄꎬｄｅｈｙｄｒａｔｉｏｎａｎｄＡＢＡｓｔｒｅｓｓ[Ｊ].Ｔｈｅｏｒ.Ａｐｐｌ.Ｇｅｎｅｔ.ꎬ２００３ꎬ１０６(５):９２３－９３０.[８]㊀ＳａｋｕｍａＹꎬＬｉｕＱꎬＤｕｂｏｕｚｅｔＪＧ.ＤＮＡ￣ｂｉｎｄｉｎｇｓｐｅｃｉｆｉｃｉｔｙｏｆｔｈｅＥＲＦ/ＡＰ２ｄｏｍａｉｎｏｆＡｒａｂｉｄｏｐｓｉｓＤＲＥＢｓꎬｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃ￣ｔｏｒｓｉｎｖｏｌｖｅｄｉｎｄｅｈｙｄｒａｔｉｏｎ￣ａｎｄｃｏｌｄ￣ｉｎｄｕｃｉｂｌｅｇｅｎｅｅｘｐｒｅｓ￣ｓｉｏｎ[Ｊ].Ｂｉｏｃｈｅｍ.Ｂｉｏｐｈｙｓ.Ｒｅｓ.Ｃｏｍｍｕｎ.ꎬ２００２ꎬ２９０(３):９９８－１００９.[９]㊀ＣｈｅｎＨꎬＬｉｕＬꎬＷａｎｇＬꎬｅｔａｌ.ＶｒＤＲＥＢ２ＡꎬａＤＲＥＢ￣ｂｉｎｄｉｎｇｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｆｒｏｍＶｉｇｎａｒａｄｉａｔａꎬｉｎｃｒｅａｓｅｄｄｒｏｕｇｈｔａｎｄｈｉｇｈ￣ｓａｌｔｔｏｌｅｒａｎｃｅｉｎｔｒａｎｓｇｅｎｉｃＡｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ[Ｊ].Ｊ.ＰｌａｎｔＲｅｓ.ꎬ２０１６ꎬ１２９(２):２６３－２７３.[１０]ＮｉｕＸꎬＨｅｌｅｎｔｊａｒｉｓＴꎬＢａｔｅＮＪ.ＭａｉｚｅＡＢＩ４ｂｉｎｄｓｃｏｕｐｌｉｎｇｅｌ￣ｅｍｅｎｔ１ｉｎａｂｓｃｉｓｉｃａｃｉｄａｎｄｓｕｇａｒｒｅｓｐｏｎｓｅｇｅｎｅｓ[Ｊ].ＰｌａｎｔＣｅｌｌꎬ２００２ꎬ１４(１０):２５６５－２５７５.[１１]ＳｕｎＳꎬＹｕＪＰꎬＣｈｅｎＦꎬｅｔａｌ.ＴＩＮＹꎬａｄｅｈｙｄｒａｔｉｏｎ￣ｒｅｓｐｏｎｓｉｖｅｅｌｅｍｅｎｔ(ＤＲＥ)￣ｂｉｎｄｉｎｇｐｒｏｔｅｉｎ￣ｌｉｋｅｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｃｏｎ￣ｎｅｃｔｉｎｇｔｈｅＤＲＥ￣ａｎｄｅｔｈｙｌｅｎｅ￣ｒｅｓｐｏｎｓｉｖｅｅｌｅｍｅｎｔ￣ｍｅｄｉａｔｅｄｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｓｉｎＡｒａｂｉｄｏｐｓｉｓ[Ｊ].Ｊ.Ｂｉｏｌ.Ｃｈｅｍ.ꎬ２００８ꎬ２８３(１０):６２６１－６２７１.[１２]ＤｏｎｇＣＪꎬＬｉｕＪＹ.ＴｈｅＡｒａｂｉｄｏｐｓｉｓＥＡＲ￣ｍｏｔｉｆ￣ｃｏｎｔａｉｎｉｎｇｐｒｏ￣ｔｅｉｎＲＡＰ２.１ｆｕｎｃｔｉｏｎｓａｓａｎａｃｔｉｖｅｔｒａｎｓｃｒｉｐｔｉｏｎａｌｒｅｐｒｅｓｓｏｒｔｏｋｅｅｐｓｔｒｅｓｓｒｅｓｐｏｎｓｅｓｕｎｄｅｒｔｉｇｈｔｃｏｎｔｒｏｌ[Ｊ].ＢＭＣＰｌａｎｔＢｉｏｌ.ꎬ２０１０ꎬ１０:４７.３１㊀第３期㊀㊀㊀㊀㊀㊀㊀李永波ꎬ等:小麦ＤＲＥＢ４蛋白的原核表达及多克隆抗体制备[１３]ＬｉｎＲＣꎬＰａｒｋＨＪꎬＷａｎｇＨＹ.ＲｏｌｅｏｆＡｒａｂｉｄｏｐｓｉｓＲＡＰ２.４ｉｎｒｅｇｕｌａｔｉｎｇｌｉｇｈｔ￣ａｎｄｅｔｈｙｌｅｎｅ￣ｍｅｄｉａｔｅｄｄｅｖｅｌｏｐｍｅｎｔａｌｐｒｏｃｅｓ￣ｓｅｓａｎｄｄｒｏｕｇｈｔｓｔｒｅｓｓｔｏｌｅｒａｎｃｅ[Ｊ].Ｍｏｌ.Ｐｌａｎｔꎬ２００８ꎬ１(１):４２－５７.[１４]ＰｅｌｌｅｇｒｉｎｅｓｃｈｉＡꎬＲｅｙｎｏｌｄｓＭꎬＰａｃｈｅｃｏＭꎬｅｔａｌ.Ｓｔｒｅｓｓ￣ｉｎ￣ｄｕｃｅｄｅｘｐｒｅｓｓｉｏｎｉｎｗｈｅａｔｏｆｔｈｅＡｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａＤＲＥＢ１Ａｇｅｎｅｄｅｌａｙｓｗａｔｅｒｓｔｒｅｓｓｓｙｍｐｔｏｍｓｕｎｄｅｒｇｒｅｅｎｈｏｕｓｅｃｏｎｄｉｔｉｏｎｓ[Ｊ].Ｇｅｎｏｍｅꎬ２００４ꎬ４７(３):４９３－５００.[１５]ＺｈｏｕＹꎬＣｈｅｎＭꎬＧｕｏＪꎬｅｔａｌ.ＯｖｅｒｅｘｐｒｅｓｓｉｏｎｏｆｓｏｙｂｅａｎＤＲＥＢ１ｅｎｈａｎｃｅｓｄｒｏｕｇｈｔｓｔｒｅｓｓｔｏｌｅｒａｎｃｅｏｆｔｒａｎｓｇｅｎｉｃｗｈｅａｔｉｎｔｈｅｆｉｅｌｄ[Ｊ].Ｊ.Ｅｘｐ.Ｂｏｔ.ꎬ２０２０ꎬ７１(６):１８４２－１８５７. [１６]刘洋洋ꎬ郭栋ꎬ楚秀生ꎬ等.转ＤＲＥＢ基因小麦新品系抗旱生理指标测定[Ｊ].山东农业科学ꎬ２０１３ꎬ４５(３):３８－４１. [１７]ＣｈｅｎＭꎬＸｕＺꎬＸｉａＬꎬｅｔａｌ.Ｃｏｌｄ￣ｉｎｄｕｃｅｄｍｏｄｕｌａｔｉｏｎａｎｄｆｕｎｃｔｉｏｎａｌａｎａｌｙｓｅｓｏｆｔｈｅＤＲＥ￣ｂｉｎｄｉｎｇｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｇｅｎｅꎬＧｍＤＲＥＢ３ꎬｉｎｓｏｙｂｅａｎ(ＧｌｙｃｉｎｅｍａｘＬ.)[Ｊ].Ｊ.Ｅｘｐ.Ｂｏｔ.ꎬ２００９ꎬ６０(１):１２１－１３５.[１８]ＣｈｅｎＭꎬＷａｎｇＱＹꎬＣｈｅｎｇＸＧꎬｅｔａｌ.ＧｍＤＲＥＢ２ꎬａｓｏｙｂｅａｎＤＲＥ￣ｂｉｎｄｉｎｇｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒꎬｃｏｎｆｅｒｒｅｄｄｒｏｕｇｈｔａｎｄｈｉｇｈ￣ｓａｌｔｔｏｌｅｒａｎｃｅｉｎｔｒａｎｓｇｅｎｉｃｐｌａｎｔｓ[Ｊ].Ｂｉｏｃｈｅｍ.Ｂｉｏｐｈｙｓ.Ｒｅｓ.Ｃｏｍｍｕｎ.ꎬ２００７ꎬ３５３(２):２９９－３０５.[１９]ＴｕＴＱꎬＶａｃｉａｘａＰꎬＬｏＴＴＭꎬｅｔａｌ.ＧｍＤＲＥＢ６ꎬａｓｏｙｂｅａｎｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒꎬｎｏｔａｂｌｙａｆｆｅｃｔｓｔｈｅｔｒａｎｓｃｒｉｐｔｉｏｎｏｆｔｈｅＮｔＰ５ＣＳａｎｄＮｔＣＬＣｇｅｎｅｓｉｎｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏｕｎｄｅｒｓａｌｔｓｔｒｅｓｓｃｏｎｄｉｔｉｏｎｓ[Ｊ].Ｓａｕｄｉ.Ｊ.Ｂｉｏｌ.Ｓｃｉ.ꎬ２０２１ꎬ２８(１２):７１７５－７１８１.[２０]倪志勇.小麦ＤＲＥＢ转录因子的分子生物学特性分析及功能鉴定[Ｄ].杨凌:西北农林科技大学ꎬ２００８.[２１]ＳａｒｋａｒＴꎬＴｈａｎｋａｐｐａｎＲꎬＭｉｓｈｒａＧＰꎬｅｔａｌ.ＡｄｖａｎｃｅｓｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｕｓｅｏｆＤＲＥＢｆｏｒｉｍｐｒｏｖｅｄａｂｉｏｔｉｃｓｔｒｅｓｓｔｏｌｅｒ￣ａｎｃｅｉｎｔｒａｎｓｇｅｎｉｃｃｒｏｐｐｌａｎｔｓ[Ｊ].ＰｈｙｓｉｏｌｏｇｙａｎｄＭｏｌｅｃｕｌａｒＢｉｏｌｏｇｙｏｆＰｌａｎｔｓꎬ２０１９ꎬ２５(６):１３２３－１３３４.[２２]ＣａｏＢꎬＨｅＸꎬＬｕｏＹꎬｅｔａｌ.Ｓａｆｅｔｙａｓｓｅｓｓｍｅｎｔｏｆｄｅｈｙｄｒａｔｉｏｎ￣ｒｅｓｐｏｎｓｉｖｅｅｌｅｍｅｎｔ￣ｂｉｎｄｉｎｇ(ＤＲＥＢ)４ｐｒｏｔｅｉｎｅｘｐｒｅｓｓｅｄｉｎＥ.ｃｏｌｉ[Ｊ].ＦｏｏｄａｎｄＣｈｅｍｉｃａｌＴｏｘｉｃｏｌｏｇｙꎬ２０１２ꎬ５０(１１):４０７７－４０８４.[２３]徐兆师.小麦抗逆相关转录因子基因的克隆与鉴定[Ｄ].北京:中国农业科学院ꎬ２００５.[２４]范玉清ꎬ刘恒ꎬ任伟.拟南芥ＤＲＥＢ１Ａ转录因子的原核表达和多克隆抗体制备[Ｊ].植物生理学通讯ꎬ２００７ꎬ４３(３):５３３－５３７.４１山东农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀第５５卷㊀。

毕赤酵母多拷贝表达载体试剂盒三叶虫(2007-4-25 13:59:55) 点击:75 回复:0 IP:60.216.104.*制作者：陈苗商汉桥毕赤酵母多拷贝表达载体试剂盒用于在含多拷贝基因的毕赤酵母菌中表达并分离重组蛋白综述：基本特征：作为真核生物，毕赤酵母具有高等真核表达系统的许多优点：如蛋白加工、折叠、翻译后修饰等。

不仅如此，操作时与E.coli及酿酒酵母同样简单。

它比杆状病毒或哺乳动物组织培养等其它真核表达系统更快捷、简单、廉价，且表达水平更高。

同为酵母，毕赤酵母具有与酿酒酵母相似的分子及遗传操作优点，且它的外源蛋白表达水平是后者的十倍以至百倍。

这些使得毕赤酵母成为非常有用的蛋白表达系统。

与酿酒酵母相似技术：许多技术可以通用：互补转化基因置换基因破坏另外，在酿酒酵母中应用的术语也可用于毕赤酵母。

例如：HIS4 基因都编码组氨酸脱氢酶；两者中基因产物有交叉互补；酿酒酵母中的一些野生型基因与毕赤酵母中的突变基因相互补，如HIS4、LEU2、ARG4、TR11、URA3 等基因在毕赤酵母中都有各自相互补的突变基因。

毕赤酵母是甲醇营养型酵母：毕赤酵母是甲醇营养型酵母，可利用甲醇作为其唯一碳源。

甲醇代谢的第一步是：醇氧化酶利用氧分子将甲醇氧化为甲醛，还有过氧化氢。

为避免过氧化氢的毒性，甲醛代谢主要在一个特殊的细胞器－过氧化物酶体－里进行，使得有毒的副产物远离细胞其余组分。

由于醇氧化酶与O2 的结合率较低，因而毕赤酵母代偿性地产生大量的酶。

而调控产生醇过氧化物酶的启动子也正是驱动外源基因在毕赤酵母中表达的启动子。

两种醇氧化酶蛋白：毕赤酵母中有两个基因编码醇氧化酶－AOX1 及AOX2。

细胞中大多数的醇氧化酶是AOX1 基因产物。

甲醇可紧密调节、诱导AOX1 基因的高水平表达，较典型的是占可溶性蛋白的30%以上。

AOX1 基因已被分离，含AOX1 启动子的质粒可用来促进编码外源蛋白的目的基因的表达。

DOI: 10.1126/science.281.5384.1860, 1860 (1998);281 Science , et al.Howard Y. Chang Adapter Protein Daxx Activation of Apoptosis Signal-Regulating Kinase 1 (ASK1) by theThis copy is for your personal, non-commercial use only.clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to othershere.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles): January 15, 2012 (this infomation is current as of The following resources related to this article are available online at/content/281/5384/1860.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/281/5384/1860.full.html#ref-list-1, 4 of which can be accessed free:cites 20 articles This article 386 article(s) on the ISI Web of Science cited by This article has been /content/281/5384/1860.full.html#related-urls 100 articles hosted by HighWire Press; see:cited by This article has been/cgi/collection/cell_biol Cell Biologysubject collections:This article appears in the following registered trademark of AAAS.is a Science 1998 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mActivation of Apoptosis Signal–Regulating Kinase 1(ASK1)by the Adapter Protein DaxxHoward Y.Chang,*Hideki Nishitoh,*Xiaolu Yang,†Hidenori Ichijo,‡David Baltimore ‡The Fas death receptor can activate the Jun NH 2-terminal kinase (JNK)pathway through the receptor-associated protein Daxx.Daxx was found to activate the JNK kinase kinase ASK1,and overexpression of a kinase-deficient ASK1mutant inhibited Fas-and Daxx-induced apoptosis and JNK activation.Fas activation induced Daxx to interact with ASK1,which consequently relieved an inhibitory intramolecular interaction between the amino-and carboxyl-termini of ASK1,activating its kinase activity.The Daxx-ASK1connection completes a signaling pathway from a cell surface death receptor to kinase cascades that modulate nuclear transcription factors.Fas is a cell surface receptor that induces apoptosis upon oligomerization (1).Fas be-longs to a family of related death receptors,including the receptors for tumor necrosis factor–␣(TNF-␣)and the cytotoxic ligand TRAIL (1,2).Fas-induced apoptosis has a critical role in maintaining peripheral im-mune tolerance (1).Fas can activate two in-dependent signaling pathways.One well-characterized pathway involves the adapter protein FADD,which recruits procaspase-8and activates a protease cascade leading to apoptosis (1,3).The second pathway is me-diated by Daxx,which can enhance Fas-in-duced apoptosis by activating the JNK kinase cascade,culminating in the phosphorylation and activation of transcription factors such as c-Jun (4,5).Because Daxx might activate JNK through a mitogen-activated protein (MAP)kinase kinase kinase (MAP3K)(6–8),we focused on ASK1.It is a MAP3K that can activate apoptosis,is activated by TNF-␣,and the dominant negative form of which can block TNF-␣Ϫinduced death (8).Us-ing an immunoprecipitation (IP)-kinase as-say after expression in human embryonic kidney 293cells (8,9),we found that ASK1activity was potentiated by coex-pression with Daxx (Fig.1A).Another MAP3K that can activate the same kinase cascade,TAK1(6),was not activated by Daxx.The Daxx domain that encodes its JNK activation and apoptotic activities (amino acids 501to 625)and fragments incorporating it (4),but not other parts of Daxx,also increased ASK1activity (Fig.1B).These data implicate ASK1as a down-stream target of Daxx.Consistent with this notion,endogenous ASK1activity was ac-tivated rapidly by Fas cross-linking in a dose-dependent manner in Jurkat cells;lowbut detectable ASK1activation was evident 5min after Fas cross-linking (Fig.1C).To determine the functional role of ASK1in Daxx and Fas signaling,we tested the effect of altering ASK1activity on the apo-ptotic activities of Daxx and Fas (Fig.2).An activated deletion mutant of Daxx,DaxxC501,can induce cell death in a Fas-independent manner in 293cells but not in HeLa cells (4).However,coexpression of ASK1and DaxxC501in HeLa cells synergistically induced apoptosis (Fig.2A).A conser-vative point mutation in the ATP binding loop of ASK1(K709R)completely abro-gated cell killing (Fig.2A).ASK1(K709M),which has less residual kinase activity than ASK1(K709R),inhibited apoptosis by Fas and DaxxC501in a dose-dependent manner (Fig.2,B and C).ASK1(K709M)also in-hibited the ability of DaxxC501and Fas to activate JNK,whereas the caspase inhibitor crmA did not (Fig.2D).Collectively,these results imply a critical role for the ASK1kinase in JNK activation and apoptosis in-duced by Fas binding of Daxx.Because MAP3Ks such as Raf directly interact with upstream signaling proteins (5),we assayed physical interaction between Daxx and ASK1by coimmunoprecipitation from transfected 293T cells.Full-length hu-man Daxx specifically coimmunoprecipitated with ASK1(Fig.3A),indicating that these two proteins physically interact in mammali-H.Y.Chang and X.Yang,Department of Biology,Massachusetts Institute of Technology,Cambridge,MA 02138,USA.H.Nishitoh and H.Ichijo,Depart-ment of Biomaterials Science,Faculty of Dentistry,Tokyo Medical and Dental University,1-5-45Yushi-ma,Bunkyo-ku,Tokyo 113-8549,and Department of Biochemistry,Cancer Institute,Tokyo,Japanese Foun-dation for Cancer Research,1-37-1Kami-Ikebukuro,Toshima-ku,Tokyo 170,Japan.D.Baltimore,Depart-ment of Biology,Massachusetts Institute of Technol-ogy,Cambridge,MA 02138,and California Institute of Technology,Pasadena,CA 91125,USA.*These authors contributed equally to this work.†Present address:Department of Molecular and Cel-lular Engineering and Institute for Human Gene Ther-apy,University of Pennsylvania,Philadelphia,PA 19104,USA.‡To whom correspondence should beaddressed.tion of ASK1.(A )Daxx activates ASK1.pcDNA3-Myc-ASK1(0.5␮g)or pCS3-Myc-TAK1(0.5␮g)was cotransfected with pEBB-Daxx (1.5␮g)into 293cells (23).ASK1and TAK1were im-munoprecipitated by anti-Myc.The immune complex was incubated with GST-MKK6and GST-SAPK/p38␥,and the kinase activity was measured with the substrate ATF2(1–109)peptide.(Top)Phosphorylation of ATF2after in vitro kinase (IVK)assay.(Bottom)Immunoblotting (WB)of immunopre-cipitated Myc-ASK1and Myc-TAK1.Fold activation of ASK1and TAK1kinase activities is indicated below.Kinase activities relative to the amount of ASK1or TAK1proteins were calculated,and the activities are shown as fold activation relative to the activities of ASK1or TAK1from Daxx-negative cells.(B )ASK1activation by Daxx deletion mutants.pcDNA3-FLAG-ASK1(0.5␮g)and each Daxx mutant (1.5␮g)were cotransfected into 293cells (left)or HeLa cells (right),and ASK1was immuno-precipitated with anti-FLAG.The immune complex was incubated with GST-MKK6,and then the kinase activity was measured with the substrate GST-SAPK/p38␥(KN).The sequences incorporated in each Daxx construct are as follows:Daxx [amino acids (aa)1to 739],Daxx ⌬C (aa 1to 625),Daxx1–501(aa 1to 501),DaxxC501(aa 501to 739),Daxx501–625(aa 501to 625),DaxxC (aa 626to 739).(Top)Phosphorylation of GST-SAPK3/p38␥(KN).(Bottom)Expression of FLAG-ASK1.Fold activation of ASK1kinase activities is indicated below.(C )Fas-induced activation of ASK1.Jurkat cells (5ϫ106)were treated with CH-11anti-human Fas (MBL,Nagoya,Japan)(100ng/ml)for the indicated times (left)or with the indicated concentrations for 30min (right).The endogenous ASK1was immunoprecipitated with anti-ASK1(DAV)(24),and the ASK1kinase activity was measured as described in (B).18SEPTEMBER 1998VOL 281SCIENCE 1860 o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o man cells.FLAG-tagged FADD was not copre-cipitated by ASK1under the same condition.To evaluate the observed Daxx-ASK1inter-action under more physiological conditions,we examined the association of endogenous Daxx and ASK1by coimmunoprecipitationin L/Fas cells,a mouse fibroblast cell line expressing murine Fas (4).Daxx became as-sociated with ASK1after Fas ligation byanFig.2.Role of ASK1in Daxx-and Fas-induced apoptosis and signaling.(A )Synthetic lethality of ASK1with DaxxC501.HeLa cells were transfected with 0.5␮g of pcDNA3-ASK1or pcDNA3-ASK1(K709R)(23)and 1.0␮g of pEBB-DaxxC501along with 0.5␮g of pCMV-lacZ reporter by calcium phosphate precipitation.Total amount of transfected DNA was made con-stant by adding vector DNA.Twenty-four hours after transfection,the cells were stained with X-Gal and scored for apoptotic morphology (4).Specific apoptosis was calculated as the percentage of apoptotic blue cells in each experimental condition minus the percentage of apoptotic blue cells (ϳ5%)in parallel vector-transfected cells.The data shown are the mean ϮSD of two to four independent experiments.(B )Inhibition of Fas-induced apopto-sis by ASK1(K709M).HeLa cells were transfected with 0.5␮g of pEBB-Fas and pCMV-lacZ and the indicated amount (in micrograms)of ASK1(K709M).Jo2antibody (12.5ng/ml)was added 16hours later.X-Gal staining was done at 24hours after transfection.Specific apoptosis was calculated as in (A).(C )Inhibition of DaxxC501-induced apoptosis by ASK1(K709M).pEBB-DaxxC501(2.0␮g)and the indicated amount (in micro-grams)of pcDNA3-ASK1(K709M)were cotransfected with 0.5␮g of pCMV-lacZ in 293cells.Twenty hours after transfection the cells were stained with X-Gal and specific apoptosis scored as in (A).(D )Inhibition of DaxxC501-and Fas-induced JNK activation by ASK1(K709M).Expression constructs for each indicated protein (1.0␮g)were cotransfected with 1.0␮g of pCMV-FLAG-JNK1in 293cells.Cells in lanes 7to 10were treated with Fas mAb (Jo2,0.5␮g/ml)for 30min before assay.JNK1was immunoprecipitated with anti-FLAG,and in vitro kinase assay with 1␮g of GST-cJun(1–79)was performed as described (4).(Top)Phospho-rylation of GST-cJun(1–79).(Bottom)Immunoblotting of immunoprecipitated FLAG-JNK1.Fig.3.Daxx interacts with ASK1.(A )As-sociation of Daxx and ASK1in 293T cells.Four micrograms of pRK5-FLAG-hDaxx,pcDNA3,or pcDNA3-Myc-ASK1(23)were cotransfected with 2.0␮g of pRK5-crmA in 293T cells by calcium phosphate precipitation.(CrmA prevents the induction of apoptosis and allows the accumulation of tranfected proteins.)After 24hours,cells were extracted in IP-lysis buffer (25),immunoprecipitated with anti-Myc coupled to agarose beads (Santa Cruz)for 3hours at 4°C,and washed three times with 500␮l of IP-lysis buffer.The IP samples as well as portions of the extracts (10%of IP input)were resolved by SDS-PAGE and immunoblotted with M2anti-FLAG (Kodak)as described (4).(B )Fas-induced interaction of Daxx and ASK1.(Left)Identification of endogenous Daxx protein in L/Fas cells.Lysate from 3ϫ107L/Fas cells was immunoprecipitated with poly-clonal anti-Daxx (DSS)(24)in the absence or presence of blocking peptide (5␮g/ml)and immunoblotted with DSS.(Right)L/Fas cells (3ϫ107)were treated with mAb Jo2(immunoglobulin G,100ng/ml)(26)for the indicated times (lanes 4to 7)or left untreated (lane 3).Cell lysates were immunoprecipitated with anti-ASK1(lanes 3to 7)and immunoblotted with DSS (top)(25).Equivalent IP of ASK1was confirmed by immunoblotting of the same membrane with anti-ASK1(bottom).(C )Recruitment of endogenous ASK1to Fas.L/Fas cells (1.5ϫ107)were incubated in the presence or absence of Jo2(2␮g/ml)for 30min at 37°C.Cells were washed once with ice-cold PBS and lysed in IP-lysis buffer.The postnuclear supernatant was immunoprecipitated with 40␮l of protein A/G-agarose (Santa Cruz)for 3hours at 4°C.In samples that were not first incubated with Jo2,isotype-matched control antibody (2␮g/ml,lane 1)or Jo2(lane 2)were added after cell lysis.Immunopre-cipitates were washed five times with lysis buffer,resolved by 7.5%SDS-PAGE,and immunoblotted for ASK1with the DAV antiserum.Positions of molecular size standards (in kilodaltons)are shown on the left.(D )Requirement of Daxx for Fas-ASK1interaction.Two micrograms of pcDNA3-ASK1(K709R),1.0␮g of pCI-AU1-hFas,and 4.0␮g of pRK5-hDaxxC (23)in the indicated combinations were transfected into 293T cells along with 2.0␮g of pRK5-crmA and vector DNA as needed to equalize total DNA.Transfected cells were extracted,immunoprecipitated with anti-AU1(Babco)and protein A/G-agarose (Santa Cruz),and immunoblotted for HA-ASK1as in (A).(E )Schematic diagram of ASK1mutants.Amino acid number of domain boundaries is indicated.pcDNA3-⌬N,⌬C,and kinase each contain a COOH-terminal hemagglutinin (HA)epitope tag.pcDNA3-ASKN contains an NH 2-terminal Myc epitope tag (23).(F )Daxx interacts with the NH 2-terminus of ASK1.Four micrograms of each ASK1mutant was cotransfected with 4.0␮g of pRK5-FLAG-hDaxx and 2.0␮g of pRK5-crmA in 293T cells.Samples:ASK1(lanes 1and 5);⌬N (lanes 2and 6);⌬C (lanes 3and 7);kinase (lanes 4and 8).Twenty-four hours after transfection cells were extracted in IP-lysis buffer and immunoprecipitated with M2anti-FLAG coupled to agarose beads (Kodak).IP samples and extract aliquots were immunoblotted by anti-HA as in (A).Positions of molecular size standards (in kilodaltons)are shown on theright. SCIENCE VOL 28118SEPTEMBER 19981861o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o magonistic monoclonal antibody (mAb);this interaction peaked after 15min and decreased thereafter (Fig.3B).The Daxx-ASK1inter-action raised the possibility that ASK1may interact indirectly with Fas through Daxx.In L/Fas cells,the endogenous ASK1was spe-cifically coimmunoprecipitated with Fas after mAb cross-linking (Fig.3C,lane 3),indicat-ing that ASK1does interact with Fas and therefore may be a component of the Fas receptor signaling complex.In contrast,ad-dition of mAb to Fas after cell lysis,which immunoprecipitates monomeric Fas (10),did not coprecipitate ASK1(Fig.3C,lane 2).The Fas-ASK1interaction is apparently mediated by Daxx because coexpression of DaxxC,the COOH-terminal 112amino acid Fas-binding domain of Daxx,blocked the Fas-ASK1in-teraction,presumably by competing out en-dogenous Daxx (Fig.3D,lane 3).The ability of DaxxC to block ASK1recruitment to Fas can explain the documented dominant nega-tive effects of DaxxC on both Fas-induced apoptosis and JNK activation (4).In the yeast two-hybrid system,ASK1interacted with Daxx but not with Fas (11),suggesting that Daxx interacts directly with ASK1and bridg-es ASK1and Fas.Deletion mutagenesis showed that the NH 2-terminal 648amino ac-ids of ASK1,termed ASKN,could interactwith Daxx (Fig.3E and F,lane 7),whereas other parts of ASK1could not interact.Deletion of the NH 2-terminal 648amino acids of ASK1,forming ASK1⌬N,caused the constitutive activation of kinase activity (12)as it does in other MAP3Ks (6).Purified recombinant glutathione S-transferase (GST)–ASKN inhibited the in vitro kinase activity of ASK1but not ASK1⌬N immunoprecipitated from cells (Fig.4A),suggesting that one or more interacting cellular factors regulate ASKN autoinhibition.ASK1⌬N exhibited constitutive cell death activity in HeLa cells in the absence of added Daxx (Fig.4B).Apoptosis induced by ASK1⌬N was quanti-tatively similar to that induced by ASK1plus DaxxC501and was not enhanced by coex-pression with DaxxC501(Fig.4B).These results indicate that an activated allele of ASK1functions as a genetic bypass of Daxx and suggests that with regard to ASK1acti-vation,the function of Daxx is to relieve the inhibition caused by the NH 2-terminal regu-latory domain.We tested this model directly by in vivo interaction assays.ASKN interact-ed with Daxx (Fig.4C,lane 2).It also spe-cifically coimmunoprecipitated ASK1⌬N (Fig.4C,lane 4),implying an intramolecular interaction in full-length ASK1.Importantly,when an excess of Daxx was coexpressedwith ASKN and ASK1⌬N,ASKN associated with Daxx but not ASK1⌬N (Fig.4C,lane 6).This supports a model whereby Daxx activates ASK1activity by displacing an in-hibitory intramolecular interaction between the NH 2-and COOH-termini of the kinase and “opening up”the kinase into an active conformation.In support of this model,ASKN can inhibit the constitutive apoptotic activity of ASK1⌬N in trans,and this inhibi-tion is fully reversed by the coexpression of Daxx (Fig.4D).The present results suggest a Fas-Daxx-ASK1axis in activating JNK and p38MAP kinase cascades.The mechanism by which ASK1is activated by Daxx is similar to that described for the activation of Byr2,a MAP3K in the Schizosaccharomyces pombe mating pheromone pathway,by its activators Ste4and Shk1(13).Fas activation has been reported to activate JNK by caspase-depen-dent (14)and -independent pathways (4,15).During apoptosis,caspases can cleave and activate PAK2and MEKK (16,17),two kinases that can activate the JNK pathway;JNK activation in this context is believed to effect morphologic changes associated with apoptosis (16).The Daxx-ASK1connection provides a mechanism for caspase-indepen-dent activation of JNK by Fas and perhaps other stimuli.In mice deficient for JNK3,hippocampal neurons are protected from ap-optosis after excitotoxic injury,illustrating that in certain circumstances JNK is essential for the apoptotic program (18).In this study,we have used several tumor-derived cell lines where JNK activation by the Fas-Daxx-ASK1axis led to apoptosis.Because FADD -deficient embryonic fibroblasts and T cells are blocked for Fas-induced apoptosis (19),at least in these cells Daxx does not provide an independent death pathway.The physiologic role of the Daxx-ASK1axis and its cell spec-ificity in vivo remain to be addressed.References and Notes1.S.Nagata,Cell 88,355(1997);A.K.Abbas,ibid.84,655(1996).2.P.Golstein,Curr.Biol.7,R750(1997).3.M.P.Boldin,T.M.Goncharov,Y.V.Goltsev,D.Wallach,Cell 85,803(1996);M.Muzio,et al.,ibid.,p.817.4.X.Yang,R.Khosravi-Far,H.Y.Chang,D.Baltimore,ibid.89,1067(1997).5.J.M.Kyriakis and J.Avruch,BioEssay 18,567(1996);C.J.Marshall,Cell 80,279(1995).6.K.Yamaguchi et al.,Science 270,2008(1995).7.L.A.Tibbles et al.,EMBO J.15,7026(1996).8.H.Ichijo et al.,Science 275,90(1997).9.293and HeLa cells were maintained in Dulbecco’s minimum essential medium (DMEM)supplemented with 10%fetal bovine serum (FBS),glucose (4.5␮g/ml),and penicillin (100U/ml)and transfected with Tfx-50(Promega).Jurkat cells were cultured in RPMI 1640medium containing 10%FBS and antibi-otics in an atmosphere of 5%CO 2at 37°C.SAPK3/p38␥and ATF2peptide (1–109)were provided by M.Goedert and Z.Yao,respectively.Cells were extracted and immunoprecipitated with Myc mAb (Ab-1,Calbiochem),FLAG mAb (M2,Kodak),or antiserum to ASK1(DAV)(19)with protein G(forFig.4.Mechanism of Daxx activation of ASK1.(A )ASKN inhibition of ASK1activity in vitro.pcDNA3-ASK1-HA orpcDNA3-ASK1⌬N-HA were transfected into 293cells and immunoprecipitated with anti-HA (12CA5)and protein A–Sepharose.Equalized input kinase activities were incubated with the indicated amount of GST or GST-ASKN for 60min at 4°C,and subjected to the immune complex kinase assay as described in Fig.1B.G-ASKN,GST-ASKN.(B )Constitutive apoptotic activity of ASK1⌬N.HeLa cells were transfected with 0.5␮g of each indicated ASK1mutant,1.0␮g of pEBB or pEBB-DaxxC501,and 0.5␮g of pCMV-lacZ reporter.Twenty-four after transfection,cells were stained with X-Gal and scored for specific apoptosis as in Fig.2A.(C )Daxx releases the COOH-terminus of ASK1from the NH 2-terminus of ASK1.293T cells were transfected with pcDNA3-Myc-ASKN,pcDNA3-ASK1⌬N,and pRK5-FLAG-hDaxx as indicated along with 2.0␮g of pRK5-crmA and vector DNA as needed.Two micrograms of each indicated DNA was transfected in lanes 1to 4;in lanes 5and 6,0.5␮g of ASKN,2.0␮g of ASK1⌬N,and 4.0␮g of Daxx were transfected.Transfected cells were extracted,immunoprecipitated with anti-Myc coupled to agarose beads,and immunoblotted with anti-HA and anti-FLAG as in Fig.3A.(D )One microgram each of pcDNA3-ASK1⌬N,pcDNA3-ASKN,and pRK5-FLAG-hDaxx was cotransfected as indicated with 0.5␮g of pCMV-lacZ and vector DNA as needed in HeLa cells.Twenty-four hours after transfection cells were stained with X-Gal and scored for specific apoptosis as in Fig.2A.18SEPTEMBER 1998VOL 281SCIENCE 1862 o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mAb-1or M2)–or protein A (for DAV)–Sepharose.Immune complex assays were performed essential-ly as described (12).Phosphorylation of ATF2pep-tide or GST-SAPK3/p38␥was analyzed by a Fuji BAS2000image analyzer.ASK1or TAK1protein was detected by immunoblotting and enhanced chemiluminescence (ECL),which in exposures less than 10min did not detect 32P radioactivity from kinase autophosphorylation.Protein levels from immunoblot were quantified by densitometry (Quantity One program,pdi).10.F.C.Kischkel et al .,EMBO J.14,5579(1995).11.EGY48yeast strain was tranformed with EG202-ASK1(K709R),pJG4-5vector,pJG4-5-mFas(192–295),or pJG4-5-mDaxx,and JK101reporter plasmids,and quantitative liquid ␤-galactosidase (␤-Gal)assay was performed (4).Relative ␤-Gal units ϮSD for ASK1(KR)alone,2.4Ϯ0.2;ASK1(KR)plus Daxx,119Ϯ8.8;ASK1(KR)plus Fas,1.8Ϯ0.4.12.M.Saitoh et al .,EMBO J.17,2596(1998).13.H.Tu,M.Barr,D.L.Dong,M.Wigler,Mol.Cell.Biol.17,5876(1997).14.M.A.Cahill et al.,Oncogene 13,2087(1996);F.Toyoshima,T.Moriguchi,E.Nishida.J.Cell Biol.139,1005(1997);S.Huang et al.,Immunity 6,739(1997).15.E.Goillot et al .,Proc.Natl.Acad.Sci.U.S.A.94,3302(1997);H.Wajant et al .,Curr.Biol.8,113(1998).16.T.Rudel and G.M.Bokoch,Science 276,1571(1997);N.Lee et al .,Proc.Natl.Acad.Sci.U.S.A.94,13642(1997).17.M.H.Cardone,G.S.Salvesen,C.Widmann,G.John-son,S.M.Frisch,Cell 90,315(1997).18.D.D.Yang et al .,Nature 389,865(1997).19.W.-C.Yeh,et al .Science 279,1954(1998);J.Zhang,D.Cado,A.Chen,N.H.Kabra,A.Winoto,Nature 392,296(1998).20.H.Hsu,J.Xiong,D.V.Goeddel,Cell 81,495(1995).21.K.Tobiume et al .,mun.239,905(1997).22.Abbreviations for the amino acid residues are as follows:A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;G,Gly;H,His;I,Ile;K,Lys;L,Leu;M,Met;N,Asn;P,Pro;Q,Gln;R,Arg;S,Ser;T,Thr;V,Val;W,Trp;and Y,Tyr.23.pEBB-Daxx (4),Daxx mutants (4),pEBB-Fas (4),pcDNA3-ASK1(8),pcDNA3-ASK1(K709R)(8),Myc-TAK1(6),pCMV-FLAG-JNK1(4),pRK5-crmA (20),EG202-ASK1(K709R)(12),and pJG4-5-mDaxx (4)were as described.ASK1⌬N,⌬C,kinase,FLAG-ASK1,Myc-ASK1,ASK1(K709M)-HA,and Myc-ASKN were constructed in pcDNA3(Invitrogen)by polymerase chain reaction (PCR).FLAG-tagged human Daxx and hDaxxC were derived from EST clone AA085057and constructed in pRK5(20)by PCR.pCI-AU1-hFas was constructed by J.Wang and M.J.Lenardo.The plas-mids of GST–human MKK6,GST SAPK3/p38␥(KN),and GST-ASKN for bacterial fusion protein were con-structed in pGEX-4T-1(Pharmacia Biotech)by PCR.24.Antiserum to ASK1(DAV)was raised to the peptide sequence DAVATSGVSTLSSTVSHDSQ,amino acids 1217to 1236in human ASK1,as described (21).Rabbit polyclonal antibody to mouse Daxx (DSS)was raised against the peptide sequence DSSTRVDSP-SHELVTSSLC (amino acids 680to 698)(22).25.293T cells (2ϫ106)[grown in DMEM supplemented with 10%FBS,penicillin-streptomycin (100U/ml),and glutamine (1mM)]were plated onto a 60-mm dish the day before transfection.Twenty-four hours after transfection,cells were washed once in ice-cold phosphate-buffered saline (PBS)and lysed in 300␮l of IP-lysis buffer [50mM Hepes (pH 7.4),1%NP-40,150mM NaCl,10%glycerol,1mM EDTA,2mM dithiothreitol]supplemented with 1mM phenyl-methylsulfonyl fluoride and 1%aprotinin.Extract (50␮l)was diluted in IP-lysis buffer (500␮l)and immu-noprecipitated with antibody reagents as described in the figure legends.In Fig.3B,L/Fas cells were lysed in 1ml of lysis buffer.Cell lysates were immunoprecipi-tated with antiserum to ASK1through use of protein A–Sepharose.The beads were washed twice with the washing buffer,separated by SDS—polyacrylamide gel electrophoresis (PAGE),and immunoblotted with anti-Daxx (DSS).26.J.Ogasawara et al .,Nature 364,806(1993).27.We are grateful to P.Svec for technical support.We thank S.Nagata,K.Matsumoto,J.Wang,M.J.Le-nardo,D.V.Goeddel,M.Goeddert,and Z.Yao for reagents,and A.Hoffmann for valuable advice and critical review of the manuscript.H.I.thanks K.Miya-zono for valuable discussion.H.Y.C.is supported by the Medical Scientist Training Program at Harvard Medical School.H.I.is supported by Grants-in-Aid forscientific research from the Ministry of Education,Science,and Culture of Japan.X.Y.is a fellow of the Leukemia Society of America.Supported by NIH grant CA51462.16March 1998;accepted 13August 1998Promotion of Dendritic Growth by CPG15,an Activity-InducedSignaling MoleculeElly Nedivi,*†Gang-Yi Wu,Hollis T.ClineActivity-independent and activity-dependent mechanisms work in concert to regulate neuronal growth,ensuring the formation of accurate synaptic con-nections.CPG15,a protein regulated by synaptic activity,functions as a cell-surface growth-promoting molecule in vivo.In Xenopus laevis ,CPG15enhanced dendritic arbor growth in projection neurons,with no effect on interneurons.CPG15controlled growth of neighboring neurons through an intercellular sig-naling mechanism that requires its glycosylphosphatidylinositol link.CPG15may represent a new class of activity-regulated,membrane-bound,growth-promoting proteins that permit exquisite spatial and temporal control of neu-ronal structure.The cpg15gene was identified in a forward genetic approach designed to isolate activity-regulated genes that mediate synaptic plasticity (1).In the adult rat,cpg15is induced in the brain by kainate (KA)and in visual cortex by light (2).During development,cpg15expres-sion is correlated with times of afferent in-growth,dendritic elaboration,and synaptogen-esis (2).Sequence analysis predicts a small,secreted protein (2)that is membrane-bound by a glycosylphosphatidylinositol (GPI)linkage (3).Antiserum generated against bacterially ex-pressed rat CPG15recognizes a protein from rat brain dentate gyrus extracts (Fig.1A)(4)of the size predicted by sequence analysis.A pro-tein of similar size is induced in Xenopus laevis after KA injections into the brain ventricle (Fig.1A)(5).In situ hybridizations using a partial clone of Xenopus cpg15indicate that the CPG15mRNA is expressed in retinal ganglion cells and in differentiated neurons throughout the central nervous system (CNS)of stage-47tadpoles (6).Xenopus CPG15protein is present in neurons and axons throughout the CNS (7,8).In the optic tectum,differentiated neurons label in a honeycomb pattern similar to N-CAM (neural cell adhesion molecule)and other cell-surface antigens,while cells in the proliferativezone have undetectable levels of CPG15(Fig.1C).To investigate the cellular function of CPG15,we used a recombinant vaccinia virus (VV)to express CPG15in optic tectal cells of albino Xenopus tadpoles (9,10).Tadpoles were infected by ventricular injection with VV car-rying rat cpg15and ␤-galactosidase (␤-gal)cDNAs in a dual promoter vector,or with a control virus containing only the ␤-gal cDNA (11).Two days after viral infection and approx-imately 24hours after the beginning of expres-sion of foreign protein (9),single tectal cells were labeled with DiI (10,12).Confocal imag-es through the entire structure of each neuron were collected at 24-hour intervals over a peri-od of 3days,and three-dimensional (3D)im-ages were reconstructed from this (13).The most prominent effect of CPG15on the morphology of tectal projection neurons was that the dendritic arbors of neurons from CPG15VV-infected animals increased their total dendritic branch length (TDBL)and be-came more complex than arbors of neurons from ␤-gal–infected or uninfected animals (Fig.2)(14).This effect was quantified as an increase in averaged TDBL (Fig.3A)and by Sholl analysis (Fig.3B).We measured the distribution of dendritic arbor sizes,expressed as TDBL,within the population of neurons from CPG15VV-infect-ed animals and from control animals (Fig.3C).All three populations of neurons showed a gradual shift toward larger TDBLs as their dendritic arbors grow.The shift toward larger TDBLs was greatest in neurons from CPG15VV-infected animals.This analysis also demonstrates the presence of a subpopulationCold Spring Harbor Laboratory,1Bungtown Road,Cold Spring Harbor,NY 11724,USA.*To whom correspondence should be addressed.E-mail:nedivi@†Present address:Department of Brain and Cognitive Sciences and Center for Learning and Memory,Mas-sachusetts Institute of Technology,Cambridge,MA 02139,USA. SCIENCE VOL 28118SEPTEMBER 19981863o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

碧云天生物技术/Beyotime Biotechnology 订货热线：400-168-3301或800-8283301 订货e-mail ：******************技术咨询：*****************网址：碧云天网站 微信公众号pCMV-mCherry-p62产品编号 产品名称 包装 D2818-1μg pCMV-mCherry-p62 1μg D2818-100μgpCMV-mCherry-p62100μg产品简介：pCMV-mCherry-p62是碧云天研发的在哺乳动物细胞中表达红色mCherry 标签的人源p62融合蛋白的质粒。

该质粒含有CMV 启动子，为卡那霉素抗性，转染后能够在靶细胞中高效表达带有红色荧光蛋白mCherry 标签的p62融合蛋白，呈现明亮的红色荧光，可以用于细胞自噬(autophagy)的研究。

本质粒转染细胞后，可以使用G418筛选稳定表达融合蛋白的细胞株。

p62也称sequestosome I (SQSTM1)，在多细胞生物(不包括植物和真菌)中高度保守，主要分布于细胞质，也可以定位于细胞核、自噬体(autophagosome)和溶酶体(lysosome)中。

p62是一种应激诱导的蛋白，可以作为信号枢纽(signaling hub)在氨基酸感应(amino acid sensing)和氧化应激等许多细胞事件中发挥重要作用，并且还可以作为PKC 、ERK1、mTORC1、NF-кB 和caspase-8等的支架蛋白(scaffold protein)而参与相应的信号转导。

p62全长440个氨基酸，包含一个PB1(Phox1/Bem1p)结构域，一个锌指结构域(ZZ)，两个核定位信号(NLS1和NLS2)，一个TB (TRAF6 binding)结构域，一个LIR 结构域(LC3-interacting region)，一个KIR 结构域(Keap1-interacting region)，和一个C 端的UBA(Ubiquitin-associated)结构域。

常见限制性‎内切酶识别‎序列(酶切位点)（BamHI‎、EcoRI‎、HindI‎I I、NdeI、XhoI等‎）Time：2009-10-22 PM 15:38Autho‎r:bioer‎Hits: 7681 times‎在分子克隆‎实验中，限制性内切‎酶是必不可‎少的工具酶‎。

无论是构建‎克隆载体还‎是表达载体‎，要根据载体‎选择合适的‎内切酶（当然，使用T 载就‎不必考虑了‎）。

先将引物设‎计好，然后添加酶‎切识别序列‎到引物5' 端。

常用的内切‎酶比如Ba‎m HI、EcoRI‎、HindI‎I I、NdeI、XhoI等‎可能你都已‎经记住了它‎们的识别序‎列，不过为了保‎险起见，还是得查证‎一下。

下面是一些‎常用的II‎型内切酶的‎识别序列，仅供参考。

先介绍一下‎什么是II‎型内切酶吧‎。

The Type II restr‎i ctio‎n syste‎m s typic‎a lly conta‎i n indiv‎i dual‎restr‎i ctio‎n enzym‎e s and modif‎i cati‎o n enzym‎e s encod‎e d by separ‎a te genes‎. The Type II restr‎i ctio‎n enzym‎e s typic‎a lly recog‎n ize speci‎f ic DNA seque‎n ces and cleav‎e at const‎a nt posit‎i ons at or close‎to that seque‎n ce to produ‎c e 5-phosp‎h ates‎and 3-hydro‎x yls. Usual‎l y they requi‎r e Mg 2+ ions as a cofac‎t or, altho‎u gh some have more exoti‎c requi‎r emen‎t s. The methy‎l tran‎s fera‎s es usual‎l y recog‎n ize the same seque‎n ce altho‎u gh some are more promi‎s cuou‎s. Three‎types‎of DNA methy‎l tran‎s fera‎s es have been found‎as part of Type II R-M syste‎m s formi‎n g eithe‎r C5-methy‎l cyto‎s ine, N4-methy‎l cyto‎s ine or N6-methy‎l aden‎i ne.酶类型识别序列ApaIType II restr‎i ctio‎nenzym‎e5'GGGCC‎^C 3'BamHI‎Type II restr‎i ctio‎nenzym‎e5' G^GATCC‎3'BglII‎Type II restr‎i ctio‎nenzym‎e5' A^GATCT‎3'EcoRI‎Type II restr‎i ctio‎nenzym‎e5' G^AATTC‎3'HindI‎I IType II restr‎i ctio‎nenzym‎e5' A^AGCTT‎3'KpnIType II restr‎i ctio‎nenzym‎e5' GGTAC‎^C 3'NcoIType II restr‎i ctio‎nenzym‎e5' C^CATGG‎3' NdeIType II restr‎i ctio‎nenzym‎e5' CA^TATG 3'NheIType II restr‎i ctio‎nenzym‎e5' G^CTAGC‎3'NotIType II restr‎i ctio‎nenzym‎e5' GC^GGCCG‎C 3'SacIType II restr‎i ctio‎nenzym‎e5' GAGCT‎^C 3'SalIType II restr‎i ctio‎nenzym‎e5' G^TCGAC‎3' SphIType II restr‎i ctio‎nenzym‎e5' GCATG‎^C 3'XbaIType II restr‎i ctio‎nenzym‎e5' T^CTAGA‎3'XhoIType II restr‎i ctio‎nenzym‎e5' C^TCGAG‎3'要查找更多‎内切酶的识‎别序列，你还可以选‎择下面几种‎方法：1. 查你所使用‎的内切酶的‎公司的目录‎或者网站；2. 用软件如：Prime‎r Premi‎e r5.0或Bio‎e dit等‎，这些软件均‎提供了内切‎酶识别序列‎的信息；3. 推荐到NE‎B的REB‎A SE数据‎库去查（网址：http://rebas‎/rebas‎e/rebas‎e.html）当你设计好‎引物，添加上了内‎切酶识别序列‎，下一步或许‎是添加保护‎碱基了，可以参考：NEB公司‎网站提供的‎关于设计P‎C R引物保‎护碱基参考‎表下载（也可见图片‎）双酶切bu‎f fer的‎选择（MBI、罗氏、NEB、Prom e‎g a、Takar‎a）再给大家推‎荐一种新的‎不需要连接‎反应的分子‎克隆方法，优点包括：①设计引物不‎必考虑选择‎什么酶切位点；②不必考虑保‎护碱基的问‎题；③不必每次都‎选择合适的‎酶来酶切质‎粒制备载体‎；④而且不需要‎D NA连接‎酶；⑤假阳性几率‎低（因为没有连‎接反应这一‎步，载体自连的‎问题没有了‎）。

人转录因子AP-1(AP-1)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。

检测范围：96T30ng/L – 1500ng/L使用目的：本试剂盒用于测定人血清、血浆及相关液体样本中转录因子AP-1(AP-1)含量。

实验原理本试剂盒应用双抗体夹心法测定标本中人转录因子AP-1(AP-1)水平。

用纯化的人转录因子AP-1(AP-1)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入转录因子AP-1(AP-1)，再与HRP标记的转录因子AP-1(AP-1)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。

TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。

颜色的深浅和样品中的转录因子AP-1(AP-1)呈正相关。

用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人转录因子AP-1(AP-1)浓度。

1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。

若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。

操作步骤1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。

2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。

在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。

加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。

3.温育：用封板膜封板后置37℃温育30分钟。

4.配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。

6.加酶：每孔加入酶标试剂50μl，空白孔除外。

7.温育：操作同3。

8.洗涤：操作同5。

Journal of Northeast Agricultural University东北农业大学学报第52卷第4期52(4):29~382021年4月April 2021西瓜PME 基因生物信息学分析及克隆王学征1,2，李钰婷1,2，张博1,2，张雨桐1,2，张晏航1,4，韩文灏3，朱子成1,2，刘识1,2，刘宏宇1,2（1.农业农村部东北地区园艺作物生物学与种质创制重点实验室，哈尔滨150030；2.东北农业大学园艺园林学院，哈尔滨150030；3.东北农业大学研究生院，哈尔滨150030；4.东北农业大学生命科学学院，哈尔滨150030）摘要：研究从拟南芥（Arabidopsis thaliana ）4个果胶甲酯酶基因和2个来自东北农业大学西甜瓜分子育种课题组西瓜转录组数据分析的果胶甲酯酶基因入手，通过BLAST 比对方法在葫芦科数据库中鉴定甜瓜（Cucumis melo L.）、西瓜（Citrullus lanatus ）果胶甲酯酶同源基因，预测分析其生物信息学。

结果表明，各级结构及理化性质与拟南芥果胶甲酯酶基因较为相似，并对其构建系统进化树，建树结果表明，葫芦科各物种间果胶甲酯酶基因家族同源性较高。

此外，利用同源克隆方法获得Cla 012554基因全长并分析该基因时空表达量，测序结果显示，克隆的Cla 012554基因序列与比对序列间总体相似度为95.62%。

实时荧光定量PCR 分析表明，Cla 012554基因在西瓜根、茎、叶和果肉中均表达，其中，茎表达量最高，根表达量最低。

同时，Cla 012554基因还可能参与西瓜果肉成熟软化。

研究为进一步探究葫芦科作物PME 基因功能提供重要参考。

关键词：果胶甲酯酶；葫芦科；生物信息学分析；表达分析中图分类号：S651文献标志码：A文章编号：1005-9369（2021）04-0029-10王学征,李钰婷,张博,等.西瓜PME 基因生物信息学分析及克隆[J].东北农业大学学报,2021,52(4):29-38.DOI ：10.19720/ki.issn.1005-9369.2021.04.004.Wang Xuezheng,Li Yuting,Zhang Bo,et al.Bioinformatics analysis and cloning of watermelon PME gene [J].Journal of Northeast Agricultural University,2021,52(4):29-38.(in Chinese with English abstract)DOI ：10.19720/ki.issn.1005-9369.2021.04.004.Bioinformatics analysis and cloning of watermelon PME gene/WANGXuezheng 1,2,LI Yuting 1,2,ZHANG Bo 1,2,ZHANG Yutong 1,2,ZHANG Yanhang 1,4,HAN Wenhao 3,ZHU Zicheng 1,2,LIU Shi 1,2,LIU Hongyu 1,2(1.Key Laboratory of Biology and Genetic Improvement Horticultural Crops (Northeast Region),Ministry of Agriculture and Rural Affairs,Harbin 150030,China;2.School of Horticulture and Landscape Architecture,Northeast Agricultural University,Harbin 150030,China;3.Graduate School of Northeast Agricultural University,Harbin 150030,China;4.School of Life Sciences,Northeast Agricultural University,Harbin 150030,China)Abstract:This study started with four pectin methylesterase genes from Arabidopsis thaliana andtwo pectin methylesterase genes from the watermelon transcriptome data analysis of the West Muskmelon Molecular Breeding Research Center of Northeast Agricultural University.The method was used to identify the pectin methylesterase homologous genes of melon (Cucumis melo L.)and watermelon (Citrullus lanatus )in the Cucurbitaceae database,and perform their bioinformatics prediction analysis.The results showed that its structure and physicochemical properties were similar to基金项目：国家自然科学基金项目（31772333）；国家西甜瓜产业技术体系项目（CARS-025）作者简介：王学征（1978-），女，教授，博士，研究方向为西瓜甜瓜遗传育种。

apmat基因序列-概述说明以及解释1.引言1.1 概述概述部分的内容如下：在现代生物学领域中，研究基因序列是一项重要的任务。

基因序列决定了生物体的遗传信息，并直接影响到生物体的性状、功能和特征。

了解基因序列的结构和功能对于理解生命的本质和生物现象的解释具有关键意义。

其中，APMAT（Apolipoprotein M-Angiopoietin-like protein 4）基因序列是近年来备受关注的一个重要研究对象。

APMAT基因是一个编码蛋白质的基因，它在人类和其他一些哺乳动物中广泛存在，并在多个生物学过程中发挥着重要作用。

APMAT基因编码的蛋白质主要参与脂代谢调控和血液中胆固醇的转运。

它的研究对于心血管疾病等许多疾病的发生和发展具有重要意义。

越来越多的研究发现，APMAT基因序列的突变和多态性与一些疾病的发病风险密切相关。

因此，深入了解APMAT基因序列的结构和功能对于揭示其在健康和疾病中的作用机制至关重要。

通过对APMAT基因序列的研究，我们可以深入了解蛋白质的生物化学特性和遗传变异对生物体的影响。

这将有助于发展新的诊断和治疗手段，为相关疾病的预防和治疗提供重要理论和实践基础。

总之，APMAT基因序列的研究是生物学领域中重要的课题之一。

了解其结构和功能将有助于我们更好地理解基因组的组成和调控机制，以及相关疾病的发生和发展。

未来的研究应深入探索APMAT基因序列的各个方面，为人类健康和疾病治疗的进一步发展提供更好的理论和实践基础。

1.2文章结构文章结构部分的内容如下：2. 正文文章的正文部分将会按照以下三个要点展开讨论。

2.1 第一个要点在第一个要点中，我们将详细介绍apmat基因序列的基本概念、结构和功能。

首先，我们会解释什么是apmat基因序列以及其在基因组中的位置和特征。

然后，我们会探讨apmat基因序列的生物学功能，比如它们在细胞分化、发育和疾病发生中的作用。

此外，我们还将介绍一些与apmat基因序列相关的研究进展和应用领域。