摘要
为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。
In order to obtain transgenic potato plants resistant to Potato virus X (PVX), Potato virus Y (PVY), Potato leaf role virus (PLRV) and Potato virus S (PVS), specific primers were designed according to the coat protein (CP) genes of PVX, PVY, PLRV and PVS, and then sub-clones of the cDNA fragments were obtained from the conserved regions of CP genes of PVX, PVY, PLRV and PVS, respectively. Then the four cDNA fragments were spliced into a chimeric cDNA. The RNA interference (RNAi) vector pBI121-pRH with the chimeric cDNA was constructed based on the vectors pHANNIBAL and pBI121. Potato transformation was conducted through agrobacterium-mediated system, and then transgenic plants obtained by tissue culture were tested for viral resistance. The fusion gene fragments RH1 and RH2 were obtained, and enzyme digestion resulted in an expected band of 1 200 bp in size, respectively. RNAi vectors containing pdk introns and RH1 and RH2 fusion genes were constructed, and BamH I/Sac I bi-enzyme was performed to obtain a fragment of about 3 200 bp in length, indicating that the RNAi vector pBI121-PRH was successfully constructed. The infusion genes were integrated into the genome of Longshu 11 by using PCR and PCR-Southern blotting. Four transgenic potato plants were proved immune to the virus infection by the virus infection resistance analysis. This study indicated that multiple virus-resistant potato varieties could be obtained by using RNAi.
作者
齐恩芳
贾小霞
刘石
陈晓艳
文国宏
胡新元
Qi Enfang;Jia Xiaoxia;Liu Shi;Chen Xiaoyan;Wen Guohong;Hu Xinyuan(Potato Research Institute,Gansu Academy of Agricultural Sciences,Lanzhou 730070,Gansu Province,China;Seed Industry Research and Development Center,Hami 839001,Xinjiang Uyghur Autonomous Region,China)
出处
《植物保护学报》
CAS
CSCD
北大核心
2019年第1期192-200,共9页
Journal of Plant Protection
基金
甘肃省农业科学院农业科技创新专项(2017GAAS39)
国家自然科学基金(31360353
31560412)
