Constructing transgenic tobacco for multiple-resistance against potato Virus Y, Cucumber Mosaic Virus and Tobacco Mosaic Virus

chinois. 利用RNA介导抗性培育抗多种植物病毒的转基因烟草。分别以马铃薯Y病毒（PVY）、烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）全长衣壳蛋白（CP）基因为模板，通过设计PCR引物和亚克隆获得PVY CP 3′端长度100 bp、TMV CP 3′端长度100 bp和CMV CP 3′端长度200 bp的cDNA片段并拼接成嵌合基因，并以此为模板构建反向重复结构嵌合基因的植物表达载体pRHPTC。将pRHPTC通过冻融法导入农杆菌LBA4404，采用叶盘法转化烟草NC89，然后测定转基因烟草对3种病毒的抗性。经卡那霉素筛选和PCR检测，共获得276株转基因烟草。Southern和Northern blot分析表明，外源基因以不同拷贝数整合于烟草基因组中；不同转基因植株中病毒RNA的积累量存在显著差异。抗病性检测显示：23%左右的转基因植株表现出对3种病毒侵染的抗性。对转基因植株扩繁后代和T1代的抗性分析表明：多病毒抗性表现稳定。利用RNA介导的抗病毒基因工程可获得同时抗多种病毒的转基因烟草，其抗病性在T0代扩繁植株和T1代植株中得到稳定遗传。

anglais. The purpose of this research was to probe the possibility of producing transgenic tobacco for multi-viral resistance via RNA-interference. Specific primers were designed according to the coat protein (CP) genes of Potato virus Y (PVY), Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV), and then sub-clones of cDNA fragments were obtained from the 3′ ends of CP gene of PVY, TMV and CMV, with length of 100 bp, 100 bp, and 200 bp, respectively. The three cDNA fragments were spliced into one chimeric cDNA. The chimeric cDNA was then introduced into the plant expressing vector pROKII in a way of inverted repeat which was supposed to form hairpin RNA (hpRNA) after transcription. The recombinant plant-expression vector pRHPTC was introduced into Agrobacterium tumefaciens LBA4404 by direct transferring, and then the target cDNA with inverted repeat was introduced into tobacco NC89. Transgenic plants obtained by tissue cultute were tested for viral resistance. The transformed tissues were selected in the presence of 100 mg•L-1 kanamycin. Two hundred and seventy-six transgenic tobacco plants were obtained and confirmed by PCR. Southern blot and Northern blot analysis of the transgenic plants demonstrated that the target chimeric DNA was integrated into tobacco genome DNA at different copy numbers and transcribed in almost all the transgenic plants. But the levels of RNA accumulation varied in different transgenic plants. Resistance assay indicated that about 23% transgenic plants were immune to the co-infection with PVY, TMV and CMV. Further tests showed the multi-viral resistance was stably inherited into T1 generation. The strategy of RNA-mediated viral resistance is feasible for producing multi-virus resistance in tobacco, and the resistance could be stably inherited into T1 generation.