Molecular Detection Of Sweet Potato Viruses In Hubei Province And Prokaryotic Expression Of Coat Protein From Sweet Potato Chlorotic Stunt Virus

Sweet potato virus diseases have been reported to widely occur in the sweet potato planting areas in the world, resulting in serious yield losses and quality deterioration of sweet potato. More than 20 viruses can infect sweet potato, mainly including Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato latent virus (SPLV), Sweet potato mild mottle virus (SPMMV), Sweet potato vein mottle virus (SPVMV), Sweet potato yellow dwarf virus (SPYDV), Sweet potato cauliflower mosaic-like virus (SP-CLV), Sweet potato leaf curl virus (SPLCV), Sweet potato virus G (SPV-G), Tobacco streak virus (TSV), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and so on. In China, SPFMV, SPLV, SPVMV, SPMMV, and SPVG are dominate and SPFMV is most widespread. Moreover, mixed infection of several viruses usually occur, especially, the mixed infection of SPFMV and SPCSV can cause more serious diseases and heavier yield losses. Recently, the occurrence of sweet potato virus diseases in Hubei Province has been increasing gradually, which becomes one of the key factors hindering the development of sweet potato industrialization. Therefore, it is very necessary to identify the main sweet potato viruses in Hubei and establish a rapid, sensitive and effective detection technique for the surveillance of viruses in the cultivation of virus-free seed tuberous root and the introduction of sweet potato.RNA viruses generate a replicative form (double-stranded RNA) during viral replication in host cells. RNA viruses can be identified through the detection of dsRNA in the infected cells, RT-PCR analysis and sequence analysis. In this study, RT-PCR was performed using anchoring random primers and dsRNA extracted from the infected sweet potato leaves collected from Ezhou City, Jingzhou City and Shiyan City in Hubei Province. Amplicons were cloned into T-vector and sequenced. Sequence analysis revealed the existence of SPCSV (SPCSV-HB), SPFMV (SPFMV-HB) and CMV in the test samples. Nucleotide sequence comparisons showed that SPCSV-HB shared more than 98% homology with the SPCSV isolates from Jiangsu, Anhui, Zhejiang, Henan, Jiangxi, Shandong, Chongqin, Sichuan, Hunan, Guangxi, and Spain, suggesting that SPCSV-HB also belongs to West African strain; SPFMV-HB had 98% homology with Japan S strain, Argentina RC-ARG isolate,92% homology with Peru EA strain, Argentina O-ARG isolate and Japan 10-0 strain.In addition, the full-length cDNA fragment of coat protein (CP) gene from SPCSV-HB and SPFMV-HB was amplified by RT-PCR, cloned into T-vector and sequenced, respectively. Sequence analysis indicated that SPCSV-HB CP gene contains 774 nucleotides enconding a peptide with 257 amino acids and predicted molecular weight of about 28.7 kDa; SPFMV-HB CP gene contains 945 nucleotides enconding a peptide with 315 amino acids and predicted molecular weight of about 35.2 kDa. Sequence alignment showed that the amino acid sequence of the coat protein of SPCSV-HB had 98-99% identities to the coat protein encoded by other isolates from Jiangsu (GenBank accession number, AGE 15456.1), Zhejiang (AGG40822.1), Shandong (AGG40813.1), Henan (AGG40820.1), Jiangxi (AGG4080808.1), Chongqin (AGS80301.1), Sichuan (AGS80283.1), Guangxi (AGG40810.1) in China, and Spain CAN 181-19 (ADA61204.1), and only 81% identities to Kenya 115-23 (ABI94408.1) and SIEA-22a (CAA09349.1) belonging to East African strain. These results further verified that SPCSV-HB, like the known isolates from other regions in Chia, belongs to West African strain. SPFMV-HB coat protein had 97-99% identities to the coat protein encoded by the other isolates from Jiangsu (AEG79937.1), Shanxi (AEG79952.1), Anhui (AEG79912.1), Guangdong (AEG79919.1), Sichuan (AET79525.1), at amino acid level.To prokaryotically expressing the coat protein of SPCSV-HB, a plasmid was constructed by inserting the BamHI/XhoI-digested PCR product of CP gene into the BamHI/XhoI-digested site of vector pET-28a in frame. After introducing with 0.5 mmol/L of IPTG, an overexpressed protein was detected in SDS-polyacrylamide gel electrophoresis and it mainly existed in inclusion body.Antisera against SPCSV-HB coat protein were prepared by immunizing rabbit three times using the purified coat protein expressed in E.coli. Western blotting demonstrated that the prepared antisera could be used to detect the SPCSV in the infected plants at a dilution ratio of 1:6000, revealing the higher specificity of the prepared antisera.