多抗马铃薯病毒及类病毒的amiRNA双元表达载体的构建及遗传转化  

Construction and Genetic Transformation of Potato Virus and Viroid Multi-resistant amiRNA Binary Expression Vectors

作　　者：牟芮 刘尚武[2] 姜丽丽[1,3] 万书明[2] 王绍鹏[2] 孙少慧 李雅南[1] 吴立萍[1] 金光辉[1] 吕典秋[2] 

机构地区：[1]黑龙江八一农垦大学农学院,大庆163319 [2]黑龙江省农业科学院植物脱毒苗木研究所,哈尔滨150086 [3]黑龙江省农业科学院博士后科研工作站,哈尔滨150086

出　　处：《分子植物育种》2017年第4期1320-1326,共7页Molecular Plant Breeding

基　　金：黑龙江省省院合作项(HZ201312);黑龙江省博士后资助经费(LBH-Z14191)共同资助

摘　　要：马铃薯病毒及类病毒病害严重影响了中国马铃薯的生产。为了培育抗病毒及类病毒的马铃薯新材料,本研究采用人工miRNA(artificial microRNA,amiRNA)策略,以马铃薯X病毒(potato virus X,PVX)与马铃薯Y病毒(potato virus Y,PVY)编码沉默抑制子P25蛋白和HC-pro蛋白的基因为靶标,利用5'RACE技术对P25基因、HC-Pro基因潜在的剪切热点进行预测。再采用overlapping PCR技术,以拟南芥mi R159a前体为骨架,设计特异性引物,构建分别靶向P25和HC-Pro基因的amiRNA表达载体。同时构建靶向马铃薯内源基因Virp1的amiRNA表达载体,将构建完成的三个amiRNA表达载体串联获得amiRNAs三联体,分别转入根癌农杆菌菌株EHA105,并用农杆菌介导法转化马铃薯品种克新13。经PCR鉴定,结果表明,目的基因片段已成功转入马铃薯中。Potato viruses and viroid disease had seriously affected the production of potato in China. In order to cultivate virus and viroids antiviral new materials of potato. In this study, we utilized the strategy of artificial miRNA （amiRNA） with the silencing suppressor P25 protein and HC-pro protein which encoded by potato viral pathogens potato virus X （PVX） and potato virus Y （PVY） as the target, and we utilized 5＇RACE technique to for- ecast the potential shear hot spots of P25 gene and HC-Pro gene. Then we used overlapping PCR technology, and also using A rabidopsis thaliana miR159a precursor as skeleton to design specific primers, so we could construct amiRNA expression vector which was respectively targeting P25 and HC-Pro genes. We constructed the amiRNA expression vector targeting the endogenous gene Virp I of potato at the same time. And we could obtain amiRNAs three by seriesing three amiRNA expression vector which were construction completed. And then we transferred into the Agrobacterium strain EHA 105 respectively, we used Agrobacterium mediated transformation of potato varieties Kexinl3. Detected by PCR, the results showed that the target gene was successfully transferred into potato.

关 键 词：马铃薯 基因沉默 联合多抗 马铃薯病毒 amiRNA 遗传转化 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]