玉米ZmWRKY41基因克隆及转录激活验证  

Cloning and transcriptional activation of ZmWRKY41 transcription Factor from maize

作　　者：李国君[1] 赵娟[1] 

机构地区：[1]山西农业大学农学院,山西太谷030801

出　　处：《山西农业大学学报（自然科学版）》2017年第6期381-389,共9页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：山西省青年科技研究基金项目(201601D202054)

摘　　要：[目的]干旱是影响作物生长发育及产量的重要因素。植物WRKY转录因子超家族在植物的生长发育、响应逆境胁迫过程中发挥着重要作用。因此,克隆分析玉米WRKY转录因子的序列特征和功能,为研究玉米耐逆分子育种提供重要抗逆基因资源。[方法]本研究以玉米自交系B73为材料提取玉米总RNA反转录cDNA,克隆、分离获得ZmWRKY41基因编码区全长序列。DNAMAN比对发现,ZmWRKY41蛋白具有保守结构域WRKYGQK和锌指结构域(zinc-finger motif)C2HC,属于第三类WRKY转录因子家族。利用生物信息学方法研究该基因蛋白质理化性质,并对其进行结构分析预测。利用PlantCARE在线工具预测、鉴定ZmWRKY41基因启动子区是否含有响应非生物胁迫的顺式作用元件。将ZmWRKY41基因编码区全长序列构建pGBKT7诱饵载体上,与GAL4DNA结合域融合,转化酵母菌株AH109验证ZmWRKY41转录因子转录激活活性。[结果]玉米ZmWRKY41基因编码区全长774bp,含有长度分别为221bp、126bp、427bp 3个外显子,共编码257个氨基酸序列。蛋白质高级结构预测发现,ZmWRKY41蛋白包含2个α-螺旋结构和5个β-折叠结构,不含跨膜结构和信号肽。ZmWRKY41基因启动子元件预测发现,该启动子中含有干旱胁迫(CGGTCA)、热胁迫(AAAAAATTTC)、低温胁迫(CCGAAA)等非生物逆境胁迫响应相关的顺式作用元件。酵母转录激活验证实验显示,将含有pGBKT7-ZmWRKY41融合表达载体转化酵母AH109菌株,能在单缺、三缺培养基正常生长且能使α-半乳糖苷酶底物分解显蓝色,表明ZmWRKY41基因具有转录激活活性。[结论]玉米ZmWRKY41基因是WRKY转录因子基因家族成员之一,在酵母体内具有转录激活活性,可能参与响应非生物逆境胁迫,为进一步研究该转录因子调控非生物逆境胁迫奠定基础。[Objective]WRKY transcription factor （TF） superfamilies play an important role in responding to abiotic stresses in plants. By analyzing WRKY TF＇s sequence characteristics and functions in maize, provide important gene resource for studying stress tolerance molecular breeding of maize. [Methods]The coding sequence of ZmWRKY41 gene was isolated from eDNA of maize inbred line B73 by PCR method. The physical and chemical properties of the WRKY protein were predicted and analyzed by bioinformatics methods. The identification of abiotic stress cis-acting elements in the ZmWRKY41 promoter region was found out by the PlantCARE online tool. The coding DNA sequence （CDS） of ZmWRKY41 gene was fused with GAL4 DNA BD of bait vector pGBKT7, to verify whether ZmWRKY41 TF in yeast strain AH109 exhibited transaetivation activity. [Results]As results, the coding DNA sequence of ZmWRKY41 was 774 bp, containing 3 of exons, which were 221 bp, 126 bp and 427 bp respectively, and encoded a polypeptide of 257 amino acid residues. The protein structure prediction found that ZmWRKY41 contained 2 α-helix and 5 β-fold, without transmembrane structure and signal peptide. ZmWRKY41 protein had a conserved domain WRKYGQK and zinc finger structure, C2HC, belonging to the third class WRKY TF family. Its promoter region contained a number of cis-acting elements that regulate and respond to abiotic stresses. Yeast transactivation activity assay suggested that ZmWRKY41 had transactivation activity. EConclusion]ZmWRKY41 gene may be involved in the response to abiotic stresses and was one of WRKY transcription factor family members with transactivation activity. And it could be acquired significant direction to study the regulation of WRKY TF on abiotic stress.

关 键 词：玉米 WRKY转录因子 非生物胁迫 酵母 转录激活 

分 类 号：S513[农业科学—作物学]