Molecular Variability Of NS2 Gene Of Rice Stripe Virus And Expressed In E.coli DH5α

Rice stripe disease caused by Rice stripe virus (RSV) is an important disease to rice. And it distributes in a large scale of China. In order to further study molecuair population genetics of RSV, here we analyzed the molecular variability of NS2 gene of 24 RSV isolates from Jiangsu, Henan, Yunnan, Shandong, Fujian provinces.The NS2 gene about 600bp was obtained by RT-PCR. Molecular variability of NS2 gene encoded by RSV RNA2 within and between 24 isolates collected from 5 provinces in China was estimated by single-strand conformation polymorphism (SSCP) and sequence analysis. The results showed that variation almost lied in all isolates from SSCP. According to sequences analysis, nucleotide identities range from 93.5% to 100%, and amio acid identities range from 93.0% to 100% of RSV NS2 gene sequence of 24 isolates from China and T, O isolates from Japan. And the genetic distance is among 0-0.065. The 26 isolates are divided into two groups by phylogenetic trees. Eight isolates which were all collected from Yunnan province shared high homology and formed group I, including Y-BS03, Y-CX02, Y-YX02, Y-BQ02, Y-HT02, Y-LQ02, Y-WD02 and Y-YR02. The other 18 isolates formed group II, including 16 isolates collected from Yunnan, Jiangsu, Henan, Shandong, Fujian provinces and T, O isolates from Japan. Between groups nucleotide identities range from 93.5% to 99.2%, and amio acid identities range from 93.0% to 98.0%. And genetic distance is among 0.038-0.065. From the phylogenetic trees by genetic distance, the isolates derived from the same region, which included Yunnan, Fujian, Shandong, Jiangsu, Henan and Japanese isolates.The isolates from the same region but different years were analyzed, and distinct varibility was showed between Yunnan province isolates. Correspondingly, we also analyzed the isolates from the same time but different regions. It showed that RSV NS2 molecular variability of 14 isolates collected in 2002 was higher than that of 10 isolates collected in 2003. It could be related to where the samples came from.In order to further understand the function of RSV NS2 in the virus life cycle, the expression vector of RSV NS2 gene was constructed and expressed in E.coli DH5a. A 49kD fusion protein product was detected by SDS-PAGE. The result of western blot showed that the fusion protein can react especially with the antiserum produced by GST-NS2 immuned to the rabbit. The second structure and the function of NS2 protein were predicted by PredictProtein (http://cubic.bioc.columbia,edu/Predictprotein/). It suggests that NS2 is a movement protein and play the role with CP, according to the reaserch of some experts.