小麦转录因子TaWRKY74-c和TaWRKY74-d基因的克隆及基于生物信息学的功能分析

Cloning and Functional Characterization of Wheat Transcription Factor TaWRKY74-c and TaWRKY74-d Genes Based on Bioinformatics Analysis

WRKY基因是近年来研究较为广泛的植物转录因子,目前许多物种中都克隆出WRKY基因。近年来,小麦中也有WRKY基因被克隆,但是由于对WRKY基因生物信息学分析不足,导致研究带有一定的盲目性。本试验以小麦品种扬麦158叶片为材料,分离了2个WRKY基因,分别编码344个和371个氨基酸,与GenBank数据库中的TaWRKY74基因高度同源,命名为TaWRKY74-c和TaWRKY74-d。蛋白质保守结构域分析表明,2个基因都含有1个WRKY保守结构域,属于Ⅲ类WRKY转录因子家族。定量PCR分析表明TaWRKY74-c和TaWRKY74-d在小麦的叶片、花和茎中均表达,且在茎中的表达量最多,在花中的表达量最少。采用Genevestigator转录组分析工具,对基因在331种环境条件(如逆境、病害、激素等刺激)、10个发育时期(如苗期、孕穗期等)和21种组织器官(如根、花、叶等)中的表达进行了分析,结果表明,该基因在小麦不同发育时期和组织器官中都有表达,且在植物遭受低温、病原体侵染等环境因子处理下,表达量发生显著改变,预示可能参与到这些生物学过程中。采用RT-PCR的方法对上述分析结果进行验证,结果表明生物学实验与生物信息学预测的结果一致。本研究将大量小麦转录组的数据应用到WRKY基因功能分析上,深化了对小麦WRKY基因家族成员功能的认识,为今后对该基因的表达分析和功能研究提供了重要线索和方向。

The WRKY proteins are broadly investigated in plants in the past twenty years. Presently, many members of this gene family have been identified and characterized in more than 10 plant species. WRKY genes were also isolated from wheat, but functional characterization of these genes is largely restricted by the lack of bioinformatic analysis. In this study we cloned the TaWRKY74-c and TaWRKY74-d genes, which shared high similarity with TaWRKY74 genes in public GenBank database and encoded transcription factors with 344 and 371 amino acids, respectively. These proteins contain highly conserved WRKY domain and belongto Class III of WRKY transcription factors family. Real-time PCR indicated that target gene expressed in flower, leaves and stem with the highest level in stem and the lowest in flower. In order to comprehensively clarify the genes functions, Genevestigator tools were used to detect genes expression patterns under 331 environmental stimuli, 10 developmental stages and 21 tissues or organs. Results showed that genes played important roles throughout plant development stages and in plant responses to cold acclimation and pathogens. We then carried out RT-PCR to verify these results. The data indicated that the expression patterns of TaWRKY74 gene under different conditions are highly consistent with bioinformatic predictions. Bioinformatic tools with some recently developed programmes in this study enriched our knowledge in wheat WRKY gene functions and provided crucial clues and information for further clarification of these genes.