大豆持绿基因GmSGR的RNA干扰载体构建及遗传转化  

RNA Interference Vector Construction and Genetic Transformation of Stay Green Gene GmSGR in Soybean

作　　者：刘阳颖 高乐 韩俊[1] 李润枝[1] 康恩宽[1] 韩天富[2] 谢皓[1] 王程[1] Liu Yangying;Gao Le;Han Jun;Li Runzhi;Kang Enkuan;Han Tianfu;Xie Hao;Wang Cheng(Key Laboratory of New Technology in Agricultural Application,College of Plant Science and Technology,Beijing University of Agriculture,Beijing,102206;Ministry of Agriculture Key Lab of Soybean Biology（Beijing）,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Bei-jing,100081)

机构地区：[1]北京农学院植物科学技术学院,北京农业应用新技术北京市重点实验室,北京102206 [2]中国农业科学院作物科学研究所,农业部北京大豆生物学重点实验室,北京100081

出　　处：《分子植物育种》2018年第17期5616-5623,共8页Molecular Plant Breeding

基　　金：国家自然科学基金(31371648); 中国博士后科学基金(2015M580154); 北京市教育委员会科研项目一般计划(KM201810020006); 北京农学院教师科研基金项目(SXQN2016104)共同资助

摘　　要：持绿基因(Stay Green gene)在植物衰老过程中起到了重要的作用。本研究利用同源序列PCR方法、Gateway重组技术和子叶节遗传转化方法,对大豆持绿基因Gm SGR进行RNAi载体的构建,并将其转入到大豆受体中,获得转基因阳性植株。结果显示,分别获得长度为786 bp的Gm SGR1 CDS序列,长度为816 bp的Gm SGR2 CDS序列及355 bp的att B-Gm SGRi干扰片段。依据RNAi引物设计原则和Gateway重组技术,将干扰片段依次导入到入门载体p DONR221和表达载体p B7GWIW2(Ⅱ)中,并转化农杆菌EHA105,通过子叶节法将其转入大豆受体中,最终获得阳性转基因植株。本研究在m RNA水平沉默同源靶基因,干扰大豆Gm SGR基因功能,结果显示其叶片和种子有趋向持绿的特性。本研究为探究持绿基因的遗传效应,作用机理提供实践参考,为高光效大豆的遗传改良,探讨植物衰老延缓机制,提高产量和品质等提供理论支持。Stay Green gene plays an important role in plant senescence.In this study,the homologous sequence PCR method,Gateway and the cotyledonary node transformation method were used to construct the RNAi vector of stay green Gm SGR and transferred into soybean receptor to obtain transgenic plants.The results showed that Gm SGR1 sequence of 786 bp in length,Gm SGR2 sequence of 816 bp in length and att B-Gm SGRi fragment of 355 bp in length were obtained respectively.According to the design principles of RNAi primers and Gateway recombination technology,the interference fragments were introduced into the intron vector p DONR221 and the inducible vector pB7GWIW2（Ⅱ） in turn,and then transformed into Agrobacterium tumefaciens EHA105,transferred into the soybean receptor through the cotyledonary node method to obtain the positive transgenic Plant.Silencing the homologous target gene at the m RNA level knocked out the function of soybean Gm SGR gene and observed that the leaves and seeds tended to be stay green.The research might provide a practical reference for exploring the genetic effect and mechanism of green gene,and provide theoretical support for the genetic improvement of high light efficiency soybean,exploring the mechanism of plant aging retardation,improving yield and quality.

关 键 词：大豆 持绿基因 基因克隆 RNA干扰载体 遗传转化 

分 类 号：S565.1[农业科学—作物学]