LeWRKY2基因的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2012.07.003

摘要/Abstract

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（rapid amplification 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。③100 μmol•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。

关键词: 番茄, 基因克隆, LeWRKY2基因, 茉莉酸

Abstract: 【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.

Key words: tomato, gene cloning, LeWRKY gene, jasmonic acid

孙清鹏, 李娜, 于涌鲲, 赵福宽, 万善霞, 潘金豹. LeWRKY2基因的克隆及功能分析[J]. 中国农业科学, 2012, 45(7): 1257-1264.

SUN Qing-Peng, LI Na, YU Yong-Kun, ZHAO Fu-Kuan, WAN Shan-Xia, PAN Jin-Bao. Molecular Cloning and Analysis of LeWRKY2 Gene[J]. Scientia Agricultura Sinica, 2012, 45(7): 1257-1264.

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