辣椒CaWRKY39基因克隆及其酵母单杂交pGADT7载体构建  

Cloning of CaWRKY39 Gene in Capsicum annuum L.and Its Yeast-One-Hybrid pGADT7 Vector Construction

作　　者：夏玉琪 郑贤涛 武士波 刘雅婷[1,3] Xia Yuqi;Zheng Xiantao;Wu Shibo;Liu Yating(College of Agriculture and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China;College of Tobacco Science,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学农学与生物技术学院,云南昆明650201 [2]云南农业大学植物保护学院,云南昆明650201 [3]云南农业大学烟草学院,云南昆明650201

出　　处：《山东农业科学》2024年第7期39-45,共7页Shandong Agricultural Sciences

基　　金：国家自然科学基金项目(32260681)。

摘　　要：辣椒是我国重要的经济作物之一,但番茄斑萎病毒(TSWV)极易造成其巨大的生产损失,亟需有效的防治策略。前期研究发现,辣椒转录因子CaWRKY39在TSWV侵染下特异性高表达,推测其可能在辣椒抗TSWV中发挥重要作用。本研究从辣椒‘萧新19’叶片中克隆了CaWRKY39基因,并对其进行生物信息学分析,结果表明,CaWRKY39的CDS全长为1 035 bp,编码一个由344个氨基酸组成、相对分子量为38 570.53 Da、理论等电点为9.59的稳定疏水蛋白,该蛋白二级结构和三级结构主要由无规则卷曲构成,占比为60.76%。系统进化分析发现CaWRKY39蛋白与棉花GhWRKY39蛋白亲缘关系最近,表明两者功能可能具有相似性。为进一步验证CaWRKY39对相关抗性基因的靶向调控作用,采用RT-PCR技术扩增CaWRKY39基因CDS序列,利用NdeⅠ、EcoRⅠ双酶切,并通过无缝克隆的方法成功构建了酵母单杂交表达载体pGADT7-CaWRKY39。本研究结果可为深入研究CaWRKY39转录因子参与辣椒抗TSWV病毒的分子机理提供技术支持和参考。Pepper(Capsicum annuum L.)is one of the most important economic crops in China,but Tomato spotted wilt virus(TSWV)has caused huge losses in its production,so effective control strategies are urgently needed.Previous research found that a WRKY transcription factor CaWRKY39 was specifically highly expressed after TSWV infection,suggesting that it may play a critical role in pepper resistant to TSWV.In this study,the CaWRKY39 gene was cloned from leaves of Xiaoxin 19 pepper.The bioinformatics analysis results showed that the full length of CaWRKY39 CDS was 1035 bp,which encoded a stable hydrophobic protein composed of 344 amino acids and with the relative molecular weight as 38570.53 Da and the theoretical isoelectric point as 9.59.The secondary structure and tertiary structure of CaWRKY39 were mainly composed of irregular curls,accounting for 60.76%.The phylogenetic analysis results indicated that CaWRKY39 had the closest genetic relationship with cotton GhWRKY39,indicating that their function might be similar.Furthermore,in order to verify the target regulating function of CaWRKY39 to related genes,the CDS of CaWRKY39 gene was cloned by RT-PCR,and the yeast-one-hybrid vector pGADT7-CaWRKY39 was successfully constructed by In-Fusion cloning with NdeⅠand EcoRⅠfor double digests.These results could provide the technical support and reference for further study on molecular mechanism of CaWRKY39 involved in the resistance to TSWV of pepper.

关 键 词：辣椒 CaWRKY39 生物信息学分析 酵母单杂交pGADT7载体构建 

分 类 号：S641.3[农业科学—蔬菜学] Q781[农业科学—园艺学]