甜菜液泡膜Na^+/H^+逆向转运蛋白BvNHX基因RNAi载体构建及遗传转化  

Construction of RNAi Vector and Genetic Transformation of BvNHX Gene of Na^+/H^+ Antiporter in Sugar Beet Tonoplast

作　　者：伍国强[1] 焦琦[1] 刘海龙[1] 蔺丽媛 Wu Guoqiang;Jiao Qi;Liu Hailong;Lin Liyuan(School of Life Science and Engineering,Lanzhou University of Technology,Lanzhou,73005)

机构地区：[1]兰州理工大学生命科学与工程学院,兰州730050

出　　处：《分子植物育种》2018年第18期5953-5961,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31460101);兰州理工大学"红柳杰出人才"培养计划项目(J201404)共同资助

摘　　要：液泡膜Na+/H+逆向转运蛋白介导的Na+区域化在植物适应盐胁迫中起着重要作用。本研究以甜菜(Beta vulgaris L.)叶片总RNA为模板,采用RT-PCR方法扩增液泡膜Na+/H+逆向转运蛋白基因BvNHX的靶片段,以中间载体pHANNIBAL和表达载体pART27为基础,采用酶切和连接的方法,构建CaMV 35S启动子驱动的含BvNHX基因片段反向重复序列的RNAi植物表达载体pARB,通过冻融法将其导入根癌农杆菌GV3101,并转入甜菜幼苗获得BvNHX-RNAi转化株系。半定量RT-PCR分析结果表明该干扰载体能特异性地导致转化植株BvNHX基因表达的沉默。这将为解析BvNHX在甜菜体内Na+区域化中的功能及其在甜菜适应盐渍生境中的作用机制提供帮助。Na＋compartmentalization mediated by tonoplast Na^＋/H^＋antiporter（NHX） plays an important role in plants adapting to salt stress. In the present study, using total RNA from the leaves of sugar beet（Beta vulgaris L.）as template, the target fragment of BvNHX gene of the tonoplast Na＋/H＋antiporter was amplified by RT-PCR.Then based on the intermediate vector pHANNIBAL and the expression vector pART27, the study constructed the plant expression vector p ARB of RNAi with inverted repeats of BvNHX gene fragment driven by the promoter CaMV 35 S by restriction enzyme digestion and ligation methods. Furthermore, the recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 by freezing and thawing method, and then it was transformed into sugar beet seedlings to obtain BvNHX-RNAi transformation lines. The results obtained by semi-quantitative RT-PCR method suggested that the interference vector could specifically lead to silence of BvNHX gene expression in transformed plants. This work could be helpful for further investigation of the roles of BvNHX in Na^＋compartmentalization and the action mechanism of its adaption to salinity environment in sugar beet.

关 键 词：甜菜 液泡膜Na^+/H^+逆向转运蛋白 RNA干扰 Na^+区域化 盐胁迫 

分 类 号：Q943.2[生物学—植物学] S566.3[农业科学—作物学]