小麦TaWRKYⅢ-A37和TaWRKYⅡc-D2基因的克隆与表达分析

Cloning and Expression Analysis of TaWRKYⅢ-A37 and TaWRKYⅡc-D2 Genes in Wheat

为了探究小麦(Triticum aestivum L.)WRKY基因的功能,采用同源克隆的方法,从小麦品种‘科农199’中克隆得到2个WRKY基因,分别命名为TaWRKYⅢ-A37和TaWRKYⅡc-D2,并对其进行生物信息学分析和不同逆境胁迫下的表达分析。生物信息学分析显示,TaWRKYⅢ-A37和TaWRKYⅡc-D2基因都含有2个内含子和3个外显子,分别编码206和138个氨基酸,编码蛋白都属于亲水性不稳定非分泌型的核蛋白。系统进化分析表明,TaWRKYⅢ-A37蛋白与其两个同源拷贝的亲缘关系最近,而TaWRKYⅡc-D2蛋白与粗山羊草亲缘关系最近。qRT-PCR结果表明,TaWRKYⅢ-A37和TaWRKYⅡc-D2基因在小麦根、茎和叶中均有表达,前者在根中表达量最高,后者在叶中表达量最高,二者均在茎中低表达;在苗期TaWRKYⅢ-A37基因受到PEG、H2O2和ABA胁迫后表达上调,NaCl处理后4~8 h内表达下调且低于对照表达水平,而且在灌浆期受到PEG、NaCl、H2O2和ABA胁迫后表达均上调;苗期TaWRKYⅡc-D2基因在NaCl、H2O2和ABA胁迫后表达下调,PEG处理2 h时表达上调且高于对照表达水平,并且在灌浆期经PEG和NaCl胁迫后表达下调,受H2O2和ABA胁迫后表达上调。该研究结果为深入探究TaWRKYⅢ-A37和TaWRKYⅡc-D2基因的抗逆功能奠定了理论基础。

In order to explore the function of wheat(Triticum aestivum L.)WRKY gene,we cloned two WRKY genes named TaWRKYⅢ-A37 and TaWRKYⅡc-D2 from wheat variety‘Kenong 199’by homologous cloning,and their bioinformatics and expression under different stresses were analyzed.Bioinformatic analysis showed that TaWRKYⅢ-A37 and TaWRKYⅡc-D2 genes contain two introns and three exons,encoding 206 and 138 amino acids,respectively.The encoded proteins are all hydrophilic unstable non secretory nucleoproteins.Phylogenetic analysis showed that TaWRKYⅢ-A37 protein was the closest to its two homologous copies,while TaWRKYⅡc-D2 protein was the closest to that of Aegilops tauschii.The results of qRT-PCR showed that TaWRKYⅢ-A37 and TaWRKYⅡc-D2 genes were expressed in the roots,stems and leaves of wheat.The former had the highest expression level in the roots,while the latter had the highest expression level in the leaves,all of which were low expression in the stems.The expression of TaWRKYⅢ-A37 gene in seedling stage was up-regulated by PEG,H2O2 and ABA stress,and down regulated within 4~8 hours after NaCl treatment,which was lower than that of the control;While the expression was up regulated by PEG,NaCl,H2O2 and ABA in the filling stage.At seedling stage,TaWRKYⅡc-D2 gene was down regulated by NaCl,H2O2 and ABA stress,and up regulated by PEG treatment for 2 hours,which was higher than that of control;While the expression was down regulated by PEG and NaCl stress at the filling stage,and up regulated by H2O2 and ABA stress.The results of this study lay a theoretical foundation for further exploring the anti-stress function of TaWRKYⅢ-A37 and TaWRKYⅡc-D2 genes.