LeWRKY2基因的克隆及功能分析  

Molecular Cloning and Analysis of LeWRKY2 Gene

作　　者：孙清鹏[1] 李娜[1] 于涌鲲[1] 赵福宽[1] 万善霞[1] 潘金豹[2] 

机构地区：[1]北京农学院生物技术学院,北京102206 [2]北京农学院植物科技学院,北京102206

出　　处：《中国农业科学》2012年第7期1257-1264,共8页Scientia Agricultura Sinica

基　　金：北京市自然科学基金项目(5102015)

摘　　要：【目的】克隆番茄WRKY2转录因子,为研究番茄抗病反应的分子机理和分子育种奠定基础。【方法】根据克隆出的番茄LeWRKY2基因特异片段(GenBank登录号:EU755368.1),运用cDNA末端快速扩增(rapidamplification of cNDA ends,RACE)技术克隆番茄LeWRKY2 cDNA全长,并用生物信息学的方法对其进行分析。用实时荧光定量PCR方法检测番茄经JA、番茄灰霉菌(Botrytis cinerea)、放线菌酮(cycloheximide)刺激时LeWRKY2基因的表达情况。【结果】①从番茄中克隆到一条全长为1 007 bp的cDNA序列,命名为LeWRKY2基因,阅读框471 bp,编码156个氨基酸。②LeWRKY2蛋白含有1个WRKY结构域和1个C2H2型锌指结构,可能具有转录、转录调控、信号传导功能。③100μmol.L-1茉莉酸(jasmonic acid,JA)刺激番茄幼苗0—60 min时,LeWRKY2基因的相对含量与JA刺激时间成正比;而刺激60—150 min时,LeWRKY2基因的相对含量与JA刺激时间成反比;④番茄灰霉菌可以诱导LeWRKY2基因的的表达,且在4 h时表达量达到最高;⑤LeWRKY2基因的转录产物的合成不依赖于蛋白质的从头合成。【结论】LeWRKY2是一种参与番茄防御反应的早期快速反应基因。【Objective】Cloning of WRKY transcription factor from tomato can provide a basis for molecular mechanism of pathogen reaction in tomato.【Method】 Using RT-PCR and RACE technology,the LeWRKY2 full length cDNA was cloned（GenBank accession： EU755368.1）,and its function was predicted by bioinformatics tools.The real-time PCR technology was used to analyze the expression levels of LeWRKY2 gene when treated with JA,Botrytis cinerea and cycloheximide.【Result】 The LeWRKY2 full length cDNA,consisted of 1 007 bp,was isolated from tomato.The bioinformatics analysis showed that it included an ORF of 471 bp.LeWRKY2 protein contained a WRKY domain and a C2H2 zinc finger motif,and it might have the function of transcription,transcriptional regulation and signal transduction.The LeWRKY2 gene expression levels were proportional to the JA treatment time in the range of 0-60 min,while in the range of 60-150 min,the LeWRKY2 gene expression levels were inversely proportional to the JA treatment time when treated with 100 μmol？L-1 JA.LeWRKY2 gene expression was induced by Botrytis cinerea,and the LeWRKY2 gene expression reached maximum abundance at 4 h.The transcript of LeWRKY2 gene was not dependent on the protein biosynthesis.【Conclusion】 LeWRKY2 gene is an immediate early gene involved in tomato defense response.

关 键 词：番茄 基因克隆 LeWRKY2基因 茉莉酸 

分 类 号：S641.2[农业科学—蔬菜学]