Chinese Wheat Mosaic Virus Movement Protein Gene Cloning, Expression And Yeast Two-hybrid Screening Study

Chinese wheat mosaic virus (CWMV) is a bipartite RNA virus which was isolated and identified by our laboratory. It is a new member of the genus Furovirus. The 3'-proximal ORF of CWMV RNA1 encodes a 37 kDa movement protein (MP) . The movement protein plays a very pivotal role in viral cell-to-cell movement. So it is important to produce an antiserum against the MP. We also seek for the genes encoding MP-associated proteins from the wheat cDNA library using yeast two-hybrid system. This work will redound to illustrate molecular mechanism about viral cell-to-cell movement.In this study, a pair of primers were designed based on the complete sequence of RNA1 of CWMV. About a 1 kb DNA fragment was amplified from the total RNA of infected wheat leaves by RT-PCR and was cloned into pGEM-T vector. The MP gene was confirmed after the 1 kb DNA fragment was sequenced. The complete MP gene was 990 nts long and encoded 229 amino acid residues. Compared with a reported CWMV MP gene sequence (EMBL: AJ012005) , the homology of nucleotide sequences was 99.6%. The MP gene was subcloned into pSBET. SDS-PAGE analysis showed that a 37 kDa protein which was equal to CWMV MP in molecular weight was highly expressed in E.coli BL21 (DE3) pLysS induced with IPTG. The MP was purified by SDS-PAGE and the antiserum against the protein was raised in mouse. Western blot and ELISA analysis showed the antiserum could combine with prokaryotic expressed protein and the titer was 1: 1200.The total RNA was isolated from wheat with Trizol and was purified into mRNA. The first-strand cDNA was synthesized from the mRNA with SMART technology, and the ds cDNA was further synthesized with 5' and 3' PCR primer by long distance PCR. The yeast strain AH109 was transformed with ds cDNA and pGADT7-Rec in order to construct the cDNA library. The library consists of 2.3×10~6 independent clones, and the titer library is 8.6×10~8 cfu/ml. About half of the clones had the inserted fragments were longer than 500 bp.The recombinant plasmid was constructed by ligating the gene of MP into pGBKT7, and then transformed into yeast Y187 to observe MP expression by western blot. As a result, the MP was expressed as a fusion protein and has immunogenic activity. The plasmid pGBKT7-MP has no toxicity to yeast and can't activate transcription of reporter genes by itself. So it can serve as a bait plasmid of yeast two-hybrid system.Y187 (MATα) can mate with AH109 (MATα) to became a AH109/Y187diploids. For this reason, Y187/pGBKT7-MP was mated with the pretransformed wheat cDNA lirary yeast strain AH 109, and the putative positive clones were obtained by nutritional selection screeing and X-a-gal assay. The yeast plasmid was isolated from the putative positive clones with lyticase method, and then the AD library inserts were amplified by PCR. The AD plasmids were rescued via transformation of E. coli, whose inserted fragments were longer than 500 bp. These putative positive clones were used to further analysis by using a modified two-hybrid system. At last, only 13 positive clones were confirmed. After sequences the positive clones were provided to the GenBank. It showed that they encoded five proteins including alternative oxidase > lipoic acid synthase-like protein -, large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase and two unknown proteins.