Complete Genomic Sequence Analysis Of Chinese Wheat Mosaic Virus (CWMV) And Prokaryotic Expression, Antiserum Production Of CWMV CP And CRP

Chinese wheat mosaic virus(CWMV, Furovirus, Virgaviridae) transmitted by Polymyxa gramins has two positive single-stranded RNAs which are packaged into two different particles. It is a serious problem affecting wheat yield and quality in winter wheat growing areas including Yantai and Weihai.To further understanding on the evolution and pathogenesis of CWMV, we determined the complete sequence and genetic diversity of CWMV isolates from Yantai and Linyi, prepared antibodies against coat protein and cystine-rich protein, and constructed infectious clone of CWMV RNA 2. The main results read as follows:Firstly, the complete genome sequences of CWMV isolates from Yantai and Linyi were determined. The RNA 1 of both isolates were 7 147 nt, while the RNA 2 of Yantai isolate was 3 563 nt and that of Linyi isolate was 3 564 nt, respectively. Both RNA1 and RNA2 contain three open reading frames(ORF). It showed that CWMV isolates were clustered in two groups according to the phylogenetic analysis based on RNA1 and RNA2 complete genome and their ORFs. Isolate Yantai was clustered to group I according to the sequences of RNA1, RNA2 and their ORFs, but not according to coat protein(CP). Linyi isolate was grouped to II. Recombination event was detected in the CP coding region of CWMV Linyi isolates.Then we prepared antiserum against coat protein(CP) and cysteine-rich protein(CRP). The CP and CRP genes of CWMV Yantai isolate were amplified by RT-PCR, ligated to prokaryotic expression vectors pEHISTEV. The resultant plasmids were transformed into the competent cells of E. coil Rosetta. When induced with IPTG, both plasmids expressed protein with molecular weight of 19 kDa. Rabbit was immunized four times with CP and CRP cut from SDS-PAGE. The titres of antisera against CWMV CP and CRP were 1:4094 and 1:2048, respectively. In the Western blot assay, the antisera showed specific positive reaction with wheat plants infected with CWMV, but not those infected with WYMV or healthy wheat plants.Finally, an infectious clone of CWMV RNA2 was constructed. The full-length RNA2 of CWMV was amplified via RT-PCR with primer containing T3 promoter, and ligated to pMD18-T to produce plasmid pMD18-T-CWMV-RNA2. This plasmid was linearized with Xba I and used as template for in vitro transcription with T3 RNA polymerase. The in vitro transcription product was mechanically inoculated to Nicotiana benthamiana leaves. Three days after inoculation, a band specific for CWMV CP was detected from the inoculated N. benthamiana leaves.