| Capsicum is an important Solanaceae vegetable crops. It is deeply loved by people, because the fruit nutrient is very rich, especially vitamin C content. However, in recent years, chilli pepper virus disease has become a major limiting factor in the development of production. Pepper vein mottle virus (Chilli veinal mottle virus, ChiVMV), one of the major viral diseases, which severely restricts the existence of the world pepper production, particularly South Korea, Malaysia, Thailand, India, Taiwan and other countries and regions. Pepper cultivation area and yield were significantly decreased. Pepper veinal mottle virus is a (+) ssRNA virus, one species of Potyviridae, Potyvirus. While the genomic structure, gene product functions, virus strains and genetic variation of Potyvirus have a comprehensive study at home and abroad, but the of research ChiVMV in these aspects is still significantly inadequate. Except India and South Korea reported two separate genome sequences, there are not any relevant reports. The putative gene functions were only deduced by compaired with other members of Potyviridae, while the molecular biology studies of ChiVMV strains differentiation and genetic variation are more rare. In this paper, total RNA extracted from Wenchang area infecting pepper leaves as a template and amplified by RT-PCR, and cloned whole genome sequence of ChiVMV-WC isolate. The structural features of the genome sequences were analyzed and forecasted by various of biological softwares, and its classification status was determined. CP gene was also cloned and expressed in E.coli DE3 cells and prepared specific antiserum for the detection of ChiVMV. The main conclusions obtained in this paper are as follows:1. ChiVMV-WC isolate genome sequence was successfully cloned. There were 9717 nt in the genome sequence, contained a 9270 nt long single open reading frame (ORF)and encoded a 350.44 kDa poly protein. As other Potyviruses, the poly-protein was digested by nine putative conserved restriction sites and produced 10 mature proteins.2. The homology of ChiVMV-WC genome sequences with the Indian and South Korean isolates (AJ237843 and AJ972878) were 85.66% and 93.54% and the amino acid sequences of coding poly-protein homology were 89.84% and 94.17% respectively. The sequence similarity of CP and 3'-UTR has been considered as an important criterion for classification of potyviruses. The comparative analysis of 3'-UTR and CP sequence of ChiVMV-WC with those of other potyviruses indicated that ChiVMV-WC is a distinct potyvirus, the homology of ChiVMV-WC and ChiVMV-VN/C7 isolates is close.3. According to genome sequences of ChiVMV a pair of primers was designed for CP gene. CP gene was amplified by PCR and cloned into the expression vectors pET-30b (+). After the reading frame is confirmed by sequencing, the recombinant plasmid pET30b-ChiVMV CP was transferred into E.coli Rosetta (DE3) and the expression of the recombinant CP protein was then induced by IPTG. SDS-PAGE analysis showed a high level expression of the recombinant CP of about 38 kDa. Expression conditions were optimized and the expression condition for CP gene expression was 0.1 mmol/L IPTG induced 2 h.4. The fusion protein was then purified with Ni2+-NTA agarose affinity chromatography, and used to immune rabbits for antiserum preparation. The optimal titer of the antiserum in indirect enzyme-linked immunosorbent assay (ID-ELISA) was determined to be 1/106. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of ChiVMV-WC. ID-ELISA testing of 20 of field samples demonstrated the sensitivity and specificity of the antiserum described in this paper.
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