单shRNA介导的对马铃薯Y病毒与烟草蚀纹病毒抗性分析-抗病毒基因工程专业论文.docx

单 单 shRNA 介导的对马铃薯 Y 病毒与烟草蚀纹病毒抗性研究 PAGE PAGE 4 RT-PCR. Sequencing results showed that TEV genome cDNA contains 9414 nucleotides (GenBank Acc. No.: EF470242) and encodes 3053 amino acids. By comparing the sequence hoMogy and the siRNA related standards, we selected nine target sequence with obvious features. Designed and synthetized nine primers, and annealled dsDNA. Insert into the pROKⅡdigested by restriction enzyme BamHⅠand KpnⅠ. The ligated products were transferred into E.coli DH5α by heat-shock. PCR and double enzymes digestion demonstrated that we successfully constructed the recombinant plant expression vectors pR-NIB-P1, pR-NIB-P2, pR-HC-P3, pR-HC-P4, pR-CI-P5, pR-CI-P6, pR-HC-P7, pR-NIB-P8 and pR-VPG-P9. The nine recombinant plant expression vectors were transferred into Agrobacterium tumfaciens EHA105. Instantaneously infect the Nicotiana benthamiana, and inoculated with the PVY isolates, then siRNA hybrid analysis.The results confirmed shRNA could express specific siRNA. The nine recombinant plant expression vectors were transferred into Agrobacterium tumfaciens LBA4404. pR-NIB-P1, pR-NIB-P2, pR-HC-P3, pR-HC-P4, pR-CI-P5, pR-CI-P6, pR-HC-P7, pR-NIB-P8 and pR-VPG-P9 were introduced into tobacco (NC89) plants via Agrobacterium tumfaciens-mediated transformation. The transformed tissues were screened in the presence of kanamycin, and the transgenic plants were confirmed by Kanr screening and PCR. We obtained 86, 106 ,104, 111, 80, 99, 114, 86 and 93 T0 generation transgenic plants with pR-NIB-P1, pR-NIB-P2, pR-HC-P3, pR-HC-P4, pR-CI-P5, pR-CI-P6, pR-HC-P7, pR-NIB-P8 and pR-VPG-P9 respectively. Transgenic plants were manually inoculated with the PVYN isolates. The result was 65.12%, 50.49%, 11.54%, 45.95%, 17.50%, 21.21%, 42.11%, 53.49%, 22.58% respectively. All the transgenic plants resistant to PVYN were asexual extender numerous, then inoculated with the TEV-SD1 isolates. The result is 41.86%, 50.94%, 7.69%, 40.54%, 15.00%, 18.18%, 28.94%, 44.