| Viral diseases of the sweet potato are highly prevalent and often cause serious damage to the plants,and severe reduction of the quality and yield of sweet potato,Cultivation of virus-free sweet potato is one of the most effective method to prevent sweet potato virus diseases and improve the production of sweet potato. Therefore,to find out the variation of sweetpotato viruses and establish a high efficient method to detect it will contribute to the integrative control of this kind of virus disease.Sweet potato feathery mottle virus(SPFMV),sweet potato latent virus (SPLV),sweet potato virus G(SPVG) and sweet potato vein mossaic virus(SPVMV) are the most predominant viral pathogens that had been isolated from sweet potato. These four viruses are all members of potyvirus,and they usually infect sweet potato with each other.Enzyme linked immunosorbent assay(ELISA) is commonly used to detect antibodies or specific antigens.But its lower sensitivity and cross-reactions between antibodies of these virus limit its specificity.It is difficult to identify different sweetpotato virus by ELISA specificially.We aimed at detecting and distinguishing these three potyviruses,SPFMV,SPLV and SPVG,simultaneously,by means of multiplex reverse transcription polymerase chain reaction(multiplex RT-PCR).For SPVMV,3' nucleotides sequence was cloned by RT-PCR and the molecular characteristics were analysed in this work,too.Three pairs of compatible primers specific for SPFMV,SPLV and SPVG,were designed in the conserved regions of the coat protein(CP) gene in this assay, according to the published nucleotide sequence.Multiplex RT-PCR was carried out and three distinct fragments were obtained,i.e.300bp,420bp and 600 bp,indicating the presence of SPFMV,SPLV and SPVG,respectively.Some essential factors that might affect the final results were analysed in the individual RT-PCR reaction,and the optimized detecting system was constructed.To investigate the molecular character of SPVMV in Henan Province,degenerate primers were designed according to published nucleotide sequences of different isolates. One clear fragment about 1800-nt was amplified by RT-PCR,which is proved to be 3' nucleotide sequence of SPVMV.Sequence results showed that this fragment contained the partial nucleotide sequence of the nuclear inclusion b(NIb),the coat protein (CP) gene,and the 3'-untranslated region(3'-UTR).The CP gene was consisted of 996 nt and encoded 332 amino acid residues.The nucleotide sequence identity and the deduced amino acid sequence identity with other isolates reported ranged from89.3% to 99.3%and from 98.5%to 95.2%,respectively.Based on the sequence results of CP gene,we can conclude that,the Henan islolate was most similar to SPVMV China (AY459611) and SPVMV Portugal(AY459604) isolates,with 98.9%and 99.3%identities respectively.And it was different from SPVMV South Africa(AY459608) isolate with 89.3%identity.CP gene was then cloned into pET-28a(+),and overexpression was performed in E.coli.SDS-PAGE showed that a 40.5-kDa fusion protein was expressed at high level. |
