| 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300 bp 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RT-PCR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。 |
| 关键词: 葡萄 异源表达 RNA干扰 RNA沉默抑制子 |
| DOI：10.11841/j.issn.1007-4333.2013.06.019 |
| 投稿时间：2013-04-17 |
| 基金项目:现代农业产业技术体系建设专项(CARS-30-bc-1) |
|
| Research on transgenic tobacco expressing p24 protein geneof Grapevine leafroll-associated virus 2 |
|
LI Jie, CHENG Yu-qin
|
| (College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China) |
| Abstract: |
| 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 HindⅢ/SacⅠ 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 SalⅠ and SacⅠ,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 T0 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. |
| Key words: exogenous expression RNA interference RNA silencing suppressor |
