The Molecular Variability Of NS3 Gene Of Rice Stripe Virus And Production Of The Antiserum

Rice stripe virus (RSV), a type member of Tenuivirus, it distributes abroad in China and has caused great decreases to rice yield. In order to further study the molecular variabiliby and the Function of NS3 protein, we detected molecular variations of NS3 gene among twenty-one isolates in china by reverse transcription-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis and clone sequencing.By RT-PCR, all the NS3 of twenty-one isolates were 630 bp in length. By sequence, these isolates and Y isolate of Yunnan and T, M isolates of Japan which have been previously reported were all 636nt .According to the sequences identity and characteristics of base variation of NS3 gene, these isolates could be divided into two groups. Besides KM isolate, eleven isolates which were all collected from Yunnan Province shared high homology and formed group I, including BSH^ SI^ DL, YLx JCH> YX and the others, nucleotide identities among these eleven isolates ranged from 95.2% to 98.7%, amio acid identities ranged from 95.3% to 99.1%. The other 23 isolates formed group II, including nine isolates collected from Fujian, Zhejiang, Jiangsu, Shanghai, Beijing, Liaoning, Henan, those are SM, ZJ, DF,JD,BH, DW, KF, YY, YY2, Y isolate of Yunnan and T, M isolates of Japan which have been reported belong to this group. It is interesting that KM of Yunnan belong to this group, too. Nucleotide identities among these isolates ranged from 96.2% to 99.4% and amio acid identities ranged from 93.9% to 100%. Between two groups, there were only 92.6-95.9% and 90.6-97.2% indentities in nucleotide and amino acid level, respectively.We also estimated the within-isolate population composition of SHE and KM isolates by SSCP analysis of 30 NS3 clones per isolate, more than 93% of the clones showed the same SSCP pattern as did the original RT-PCR product and 6% of the clones SSCP pattern are different. These support the hypothesis that each isolate had one predominant sequence variant and our results showed also that these RSV isolates had a qusispecies structure.The expression vector of RSV NS3 gene is constructed, a 49kDa fusion protein product is expressed in Escherichia coll BL21 successfully. The result ofwestern blot shows that GST-NS3 can reacts with the antiserum of RSV and GST-NS3 has the same immunocompetence as the natural protein of RSV. The antiserum of GST-NS3 is produced and is used in the detection of RSV by ELISA, the sensitivity is Img fresh leaf.