Aptamer And SiRNA Mediated Transgenic Resistance And Expression Of Tobacco Vein Banding Mosaic Virus Proteins

Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Potato virus X (PVX) are the most important viruses in the tobacco plants. The mixed infection of viruses is very common, and it caused more severe losses in tobacco production. In this paper, we obtained multiple virus resistant transgenic plants using two different strategies: aptamer and siRNA. Tobacco vein banding mosaic virus (TVBMV) is one species of the genus Potyvirus. The losses caused by TVBMV had been increasing during the recent years. In this paper, we obtained the complete genomic sequence of TVBMV from Yunnan China and have nine proteins of TVBMV expressed in the E. coli BL21 (DE3), and then prepared the antiserum against TVBMV CP. The detailed results read as follows:The random nonapeptides specifically binding to the CP of CMV, the N-terminal 83 amino acids of the HC-Pro interacted with PVY HC-Pro and GFP genes were simultaneously cloned to the pMD18-T vector, resulting in the plasmid pMD18 C-P-GFP. Three mutants were obtained by site-directed mutagenesis. We cloned C-P-GFP and these three mutants to the plant expression vector pROKⅡand screened by agroinfiltration the most efficient resistance inducing vector, which was introduced into tobacco plants (K326) via agrobacterium tumefaciens-mediated transformation. After tissue culture, we obtained 15 regeneration plants that were PCR-positive. After inoculation, one regeneration plant showed resistance to PVY, three to CMV, and one to both PVY and CMV. We first obtained multiple virus resistance transgenic tobacco plants using aptamer strategy.The most effective siRNA and region degraded PVX CP gene, CMV CP gene and PVY HC-Pro gene were screened using software and then constructed the reverse repeat plant expression vector. The vector was introduced into tobacco plants (K326) via agrobacterium tumefaciens-mediated transformation. Among the 22 regeneration plants, two were resistant to PVY, eight resistant to CMV, six resistant to PVX, and one resistant to PVY, CMV and PVX together. ELISA and real-time RT-PCR showed that virus accumulation in transgenic plants was lower than that in the susceptible control. This indicated that screened effective siRNA could degrade virus mRNA specifically and restrain virus replication.The complete genomic sequence of TVBMV-YND, an isolate from Yunnan, was determined by sequencing overlapping cDNA fragments obtained by RT-PCR with degenerate and/or specific primers. The genome is composed of 9,570 nucleotides (nt) excluding the 3′-terminal poly(A) tail and contains one single open reading frame of 9,240 nt encoding a large polyprotein of 3,079 amino acids with predicted Mr of 348.6 kDa. TVBMV-YND had a rare Q/N cleavage site for NIb/CP and uncommon RITC motif in HC-Pro that is crucial for aphid transmission of potyviruses.The CP gene of TVBMV YND was cloned into expression vector pET22b(+) and transferred into E.coli BL21(DE3). SDS-PAGE showed TVBMV CP gene was expressed as a 35.0 kDa fusion protein to high level when induced with IPTG. Antiserum against TVBMV CP obtained with the fusion protein, and resulting titer was 1:4 096 by ELISA. Western blotting and dot-immunobinding assay (DIBA) results showed that the acquired antiserum could specifically react with both TVBMV CP expressed in E.coli and extracts from diseased plants. Nine, except P3, of 10 TVBMV proteins were successfully expressed in E.coli. Solubility analysis showed that the soluble protein in the HC-Pro, NIa-VPg and CP proteins were suitable for purification, while others weren't because the soluble parts were lower than 10%. Structures prediction of TVBMV proteins using bioinformatics software showed that 6K2 and P3 were transmembrane proteins while others were not. All the results are useful to the crystalization and structure/function researches of TVBMV proteins.