羊草WRKY40的克隆及其转基因烟草抗病性分析  

Cloning of WRKY40 Gene from Leymus chinensis and Analysis of Disease Resistance in Transgenic Tobacco

作　　者：李丹妮 刘佳丽 张继涛[2] 顾宝祥 朱凤金 管清杰 LI Danni;LIU Jiali;ZHANG Jitao;GU Baoxiang;ZHU Fengjin;GUAN Qingjie(Key Laboratory of Restoration and Reconstruction of Northeast Saline-alkali Vegetation,Ministry of Education,College of Life Sciences,Northeast Forestry University,Harbin 150040;North-east Institute of Geography and Agroecology,Chinese Academy of Sciences,Changchun 130000)

机构地区：[1]东北盐碱植被恢复与重建教育部重点实验室,东北林业大学生命科学学院,哈尔滨150040 [2]中国科学院东北地理与农业生态研究所,长春130000

出　　处：《植物研究》2023年第3期412-420,共9页Bulletin of Botanical Research

基　　金：国家自然科学基金项目(32171989);科技基础资源调查专项(2021FY100403)。

摘　　要：为了培育抗白叶枯病菌(Xanthomonas oryzae pv.oryzae)、纹枯病菌(Rhizoctonia solani)、稻瘟病菌(Magnaporthe oryzae)以及恶苗病菌(Fusarium fujikuroi)等病菌的水稻品种,通过挖掘抗病基因选育抗病品种。利用RT-PCR方法从羊草(Leymus chinensis)叶片中克隆LcWRKY40基因(登录号:MN187915),其CDS全长1053 bp,编码350个氨基酸,分子质量为38.1 kDa。生物信息学分析结果表明:LcWRKY40一级结构包含WRKY结构域,并且存在锌指蛋白结构域以及核定位序列。系统进化树的构建及基序分析发现LcWRKY40和HvWRKY38亲缘关系接近。烟草(Nicotiana tabacum)亚细胞定位结果表明LcWRKY40蛋白定位于细胞核中,验证了软件预测。经qRT-PCR组织特异性表达分析表明LcWRKY40基因在羊草的根、茎、叶、叶鞘、稃和花药中都有表达,但表达量在叶中最高,稃中最低。利用水稻的白叶枯病菌、纹枯病菌、稻瘟病菌以及恶苗病菌对转基因LcWRKY40烟草植株与野生型烟草植株进行接菌试验,研究结果表明转基因LcWRKY40烟草植株对4种病菌有不同程度的缓解作用,对稻瘟病菌与恶苗病菌的侵害表现出较高抗性。因此推测LcWRKY40蛋白在抵御病害胁迫、提高植物病原菌抵抗力等信号途径中发挥关键调节作用。In order to cultivate rice varieties resistant to Xanthomonas oryzae pv.oryzae,Rhizoctonia solani,Magnaporthe oryzae and Fusarium fujikuroi,the method of mining resistance genes is an important way to select resistant varieties.RT-PCR method was used to clone the LcWRKY40 gene(MN187915)from the leaves of Leymus chinensis.The result showed that the CDS was 1053 bp in length and encoded 350 amino acids with a molecular weight of 38.1 kDa.The results of bioinformatics analysis showed that the primary structure of LcWRKY40 contained WRKY domain,zinc finger protein domain and nuclear localization sequence.Phylogenetic tree construction and motif analysis showed that the phylogenetic relationship between LcWRKY40 and HvWRKY38 was closed.The results of subcellular localization in tobacco showed that the LcWRKY40 protein was located in the nucleus,verified by the software prediction.qRT-PCR tissue specific expression analysis showed that LcWRKY40 gene was expressed in root,stem,leaf,leaf sheath,Lemma and anther of Leymus chinensis respectively,but the expression level was the highest in leaf but the lowest in Lemma.Transgenic LcWRKY40 tobacco plants and wild-type tobacco plants were inoculated with X.oryzae pv.Oryzae,R.solani,M.oryzae and F.fujikuroi,respectively,which showed that transgenic LcWRKY40 tobacco plants could alleviate the four pathogens in different degrees,and showed high resistance to M.oryzae and F.fujikuroi.Therefore,it was speculated that LcWRKY40 protein played a key regulatory role in signal pathways such as resisting disease stress and improved the resistance of plant pathogens,which layed a molecular foundation for the study of LcWRKY40 function and abiotic stress.

关 键 词：羊草 LcWRKY40基因 转基因烟草 抗病性 

分 类 号：S544[农业科学—作物学]