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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 full-length sequence of this gene was amplified by PCR from the T vector (p-G2-p24) containing the p24 gene of Grapevine leafroll-associated virus 2 (GLRaV-2) ’End length of 300bp positive and negative segments. The p24 protein gene full-length sequence was cloned into the expression vector pCsuper 1300+ to obtain a recombinant plasmid pCsuper1300-p24. The forward and reverse fragments were successively inserted into the intron-containing intermediate vector pBSint to obtain the recombinant intermediate vector pBSint-p24-F-R. Then pBSint-p24-FR was double-digested with HindIII and SacI, and the tandem intron sequence of the cut intron was cloned into the plant expression vector pCsuper 1300+ to obtain the RNAi recombinant plasmid pCsuper- p24-FR. PSuper1300-p24 and pCsuper-p24-F-R were transformed into Nicotiana benthamiana by Agrobacterium tumefaciens invasion leaf disc method respectively. After RTPCR and PCR detection, 34 and 12 positive T0 and T1 positive lines were obtained respectively, and 17 and 7 positive lines of T0 and T1 were transformed with pCsuper-p24-F-R respectively. The results provide experimental materials for the study of p24 function and cultivation of new RNAi-mediated anti-virus grape germplasms.