Cloning,Sequencing And Overexpression In E.coli Of Coat Protein Gene Of Sweet Potato Virus G

Sweet potato which has a larger growth area in china is one of important crops,feedingstuff and material of industry. Its fertility and wide adaptation make this crop stand in an important position with the increasing need of provision. But the sweet potato virus diseases severely damage its production and quality. While up to now there is no effective chemicals, the most effective way to prevent virus diseases is to grow the virus-free sweet potato by the technology of stalk meristem culture. So the differentiation of sweet potato viruses and the preparation of specific antiserum and establishing of the technology of viruses detection are the keys to virus-free sweet potato. Sweet Potato Virus G usually infects Sweet Potato with Sweet Potato feathery mottle virus and some other potyvirus. As a result it severly destroies the production of sweet potato ,for example the reduction of production and quality of sweet potato and degradation of variety.So far Sweet Potato Virus G is deficient in molecular biology,also there is no research about SPVG in China. This research mainly cloned, sequenced the CP gene of SPVG-HN and found the divergence with other strains of SPVG .Finally we prepared antiserum for SPVG. by overexpressing the CP gene in E. coli.It will lay the basis of the fast detection technology of SPVG.According to published nucleotide sequence of coat protein (CP) gene of Sweet Potato Virus G(SPVG),a pair of special primers were designed and synthesized. With viral RNA extracted from the leaves of Ipomoea setosa infected by SPVG, a double-strand cDNA fragment of CP gene of SPVG-HN was obtained by RT-PCR amplification. The fragment was cloned into pMD18-T vector and sequenced. The results of sequence analysis showed that the CP gene consisted of 1065 nt encoding 355 amino acid residues. The CP gene nucleotide sequence was about 98% identical to Egyptl(AJ515380), LSU-1(AY178991), LSU-3(AY178990) and SPVG-CH (X76944) isolates, respectively, and was 85.5% identical to SPVG-CH2 (Z83314) isolated from Guangdong province.It displays SPVG-HN is more relative to SPVG-CH than to SPVG-CH2. Then the CP gene was constructed into expression vector pET30a ( + ) for overexpression in prokaryotic cells. The result of SDS-PAGE...