葡萄卷叶伴随病毒2号p24蛋白基因转化烟草的研究  

Research on transgenic tobacco expressing p24 protein gene of Grapevine leafroll-associated virus 2

作　　者：李捷[1] 程玉琴[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193

出　　处：《中国农业大学学报》2013年第6期138-142,共5页Journal of China Agricultural University

基　　金：现代农业产业技术体系建设专项(CARS-30-bc-1)

摘　　要：以含有葡萄卷叶伴随病毒2号(Grapevine leafroll-associated virus 2,GLRaV-2)p24蛋白基因的T载体(p-G2-p24)为模板,PCR扩增该蛋白基因的全长序列及其5′端长300bp的正反向片段。将p24蛋白基因全长序列定向克隆到表达载体pCsuper 1300+上,得到重组质粒pCsuper1300-p24。将正反向片段先后插入具内含子的中间载体pBSint上,得到重组的中间载体pBSint-p24-F-R。然后用HindⅢ和SacⅠ双酶切pBSint-p24-F-R,将切下的带内含子的正反向串联片段定向克隆到植物表达载体pCsuper 1300+上,得到含有发夹结构的RNAi重组质粒pCsuper-p24-F-R。将pSuper1300-p24和pCsuper-p24-F-R分别通过农杆菌浸润叶盘法转化本生烟。经过RTPCR和PCR检测,分别获得了异源表达p24的T0和T1代阳性株系各34和12个,转化pCsuper-p24-F-R的T0和T1代阳性株系各17和7个。该结果可为p24功能研究及培育RNAi介导的抗病毒葡萄新种质提供试验材料。The vector p-G2-p24 contained Grapevine leafroll-associated virus 2（GLRaV-2） p24 protein gene was used to amplify sense/anti-sense strands （300 bp in size） and full sequence of p24 protein gene, respectively. PCR products of complete sequence of p24 protein gene were digested with Hind Ⅲ/Sac I and cloned to the expression vector pCsuper 1300 ＋ to obtain recombinant vector pCsuper1300-p24, The positive sense and anti-sense strands were separately inserted into the middle vector pBSint which contains an intron, to produce pBSint-p24-F-R. The obtained recombinant middle vector pBSint-p24-F-R was digested with Sal I and Sac I , and the restricted products were cloned into the expression vector pCsuper 1300 ＋ to construct the ihpRNA vector pCsuper-p24-F-R. The pCsuper1300- p24 and pCsuper-p24-F-R were transformed into Nicotiana benthamiana mediated by Agrobacterium tumefaciens, respectively. PCR and RT-PCR testing showed that 34 To and 12 T1 positive lines expressing p24 ,and 17 T0 and 7 T1 lines that transformed with the pCsuper1300-p24-F-R were obtained, respectively. These results could provide experimental materials for the function study of p24 and creation of grapevine germplasms with resistance to viruses by RNAi technology.

关 键 词：葡萄 异源表达 RNA干扰 RNA沉默抑制子 

分 类 号：S663.1[农业科学—果树学]