Construction Of Potato Tetravalent RNAi Plant Expression Vector And Its Potato Research

Potato is susceptible to many viruses during the growing period. Like potato germplasm deterioration, quantity decrease and quality degradation which could be caused by virus infection.PVX,PVY,PLRV and PVS infections are the important reasons for the degradation of potato in our country,seriously poses harm to the production of potato,In particular,the loss caused by the mixed infection of 2 or more kinds of viruses is far greater than the single infection of the virus.It is an important way for plant anti-virus gene engineered with virus genes to obtain transgenic plants. Traditional methods are difficult to obtain virus immune plant, and RNAi can effectively prevent the virus invasion which is a more effective and safe way to improve the resistance of the virus. In this study,RNA interference technology was used, and we constructed the fusion gene with fragments of four virus coat protein gene, and then constructed the expression vector of the fusion gene with reverse repeated sequence,at last anti PVS,PVY,PLRV and PVX transgenic potato was obtained by agrobacterium mediated transformation. The main results obtained in this study are as follows:1.Cloning of coat protein genes of PVX,PVS,PVY and PLRV:With NCBI PVX(Z34261.1), PVS(GU319954.1), PVY(X54611.1) and PLRV(AY307123.1) reported coat protein gene sequence,primers designed through Primer premier 5.0. Clones of PVX(714bp),PVS(885bp),PVY(804bp) and PLRV(627bp) four virus coat protein gene obtained. Compared with the reported sequences,the homology is more than 96%.2.Fusion of PVX-CP,PVY-CP,PLRV-CP and PVS-CP gene fragments:Using the biological analysis software RNA structure to analyze the folding and four kind of virus m RNA,screening the complementary si RNA affinity highest site. The region of PVX-CP gene 300 bp,PVS-CP gene 255 bp,PVY-CP gene 300 bp and PLRV-CP gene 299 bp was selected as the RNA interference region,Overlap-PCR CP gene fragment of four virus in accordance with the mutual splicing method to construct a forward and reverse XSYV-rh fusion gene and X11SYV11-rh fusion gene.3.RNAi vector construction:The intermediate vector p RH contains the forward fusion gene XSYV-rh and the reverse fusion gene X11SYV11-rh was constructed. The plant expression vector containing " X11SYV11-rh-INTRON-XSYV-rh " was constructed by the method of enzyme digestion and then the plant expression vector p BI121-p RH was constructed.And it was successfully introduced into Agrobacterium LBA4404.4.Transformation of potato:Carrying plants p BI121-p RH expression vectors for Agrobacterium tumefaciens of Longshu No.3,Longshu No.10 and Longshu No.11 three susceptible virus excellent potato varieties by genetic transformation.Longshu No.11 were obtained with 20 resistant plants by PCR detection of 6 positive strains.