Molecular Detection,Cloning Of Genomic Sequence And Expression Of Coat Protein(CP) Gene Of Sweet Potato Badnavirus

Sweet potato Badnavirus(SPBV),is double-stranded DNA virus infected sweet potato recently found,including Sweet potato Badnavirus A(SPBV-A)and Sweet potato Badnavirus B(SPBV-B).There is few current research reports on SPBV,the distribution and Molecular characterization of SPBV is unclear.In this study,sweet potato viruses samples collected from the country's main production areas were detected,and partial genome sequences of the two viruses were sequenced,and high-potentially expressed the coat protein(CP)gene of SPBV-B in Rosetta.This research lay the foundation for the development of rapid check-test technology and comprehensive prevention and control of SPBV.According to the nucleotide sequence reported by Gen Bank of SPBV-A and SPBV-B,the primers were designed.200 sweet potato samples collected from 16 provinces and municipalities of China was tested.Sweet potato leaves total DNA were taken as mould,then PCR was carried out and nucleus sequence was to be measured next.Results showed that,there are 30 samples detected SPBV-A,the detection rate is 15%,and the distribution of these samples is from Jilin,and Shandong,Hebei,Shanxi,Shaanxi,Henan,Anhui,Guangxi,Fujian,Jiangsu,Hunan,Chongqing,Sichuan provinces and cities.there are 35 samples detected SPBV-B,the detection rate is 17.5%,the samples located in Jilin,Shandong,Hebei,Henan,Anhui,Shanxi,Shaanxi,Fujian,Jiangsu,Hunan,Chongqing,Sichuan provinces and municipalities.Especially,this is first reported that the virus infected the sweet potato in China.According to nucleotide sequence reported of SPBV-A and SPBV-B genome,the overlap primer was designed.Sweet potato leaves that carried SPBV total DNA were taken as mould,and polymerase chain reaction(PCR)and nucleotide sequencing were used for cloning.Results for sequence analysis showed that,about 2.2 kb of SPBV-A genome sequence were obtained and the nucleotide sequence identity is 86%,including hypothetical protein gene and partial polyprotein gene.About 5.1 kb of SPBV-B genome sequence were obtained and the nucleotide sequence identity is 94%,including two partial polyprotein genes.According to SPBV-A and SPBV-B nucleotide sequence which was obtained,the primers were designed.Furthermore,the region of 5'-terminal 840 nts of SPBV-A coat protein(CP)gene and the region of 3'-terminal 798 nts of SPBV-B coat protein(CP)gene were obtained by PCR,and cloned into BamH?and Hind ? restriction site of expression vector pET-28a(+)for over-expression in Rosetta.Furthermore,the CP gene prokaryotic expression vector builded triumphantly,and the SDS-PAGE showed that about 36 kD specific fusion protein was produced after induction by IPTG.The antibody against the expressed SPBV-B CP infected maize leaves.