Function Reseach Of Virus-derived Small Interfering RNAs

Rice grassy stunt virus (RGSV) and rice ragged stunt virus (RRSV), two kinds of important rice virus, spread by the same insect-Nilaparvata lugens, and caused severe damage to rice production in southeast Asia. Some studies show that the virus-derived small interfering RNA play an important role in disease symptom formation mechanism. Our study focus in a interesting phenomenon that the rice infected by RGSV or RRSV will accumulate large amounts of virus-derived small interfering RNA in vivo. We speculate that these vsiRNAs may target and cleave the rice endogenous gene and participate in virus disease or symptom formation, thus speculate the vsiRNAs exist some potential function in the process of virus.A large number of vsiRNAs were gotten by analysis of Small RNA high-throughput sequencing which infected with RGSV and RRSV of Nipponbare rice in this experiment. According to the difference between the abundance value of these vsiRNAs and the base’s preferences of AGO protein combining with siRNA in RNAi pathway of rice, the vsiRNA which may have potential function were screened out. While, the target gene of these vsiRNAs in rice plants were made prediction. We detect the expression of these target genes in vivo of disease and healthy plants by real-time PCR. Results showed that relative expression of six target genes are showing a downward trend. The target gene Os04g30420 of RRSV-siRNA 391 and Os01g54119 of RRSV-siRNA 401 shows a obvious down-regulation. The target gene Os04g51180.2 of RGSV-siRNA 214 and Os04g08340.1 of RGSV-siRNA 233 have a down-regulation but not obvious. The target gene Os09g20010.2 of RGSV-siRNA 217 and Os03g62650 of RRSV-siRNA 391 are only showed a trend of downward. We preliminarily verified that the relative expression of endogenous target gene which targeted by vsiRNAs in rice exist down-regulation.In order to verify the the target genes which have a expression down-regulation in disease and healthy plants relate with the existence of vsiRNA or not. We have constructed RGSV-siRNA214、RGSV-siRNA215、RGSV-siRNA217、 RGSV-siRNA233、RRSV-siRNA305、RRSV-siRNA391 and RRSV-siRNA401 over-expression vectors and obtain corresponding transgenic rice plants by the method of rice genetic transformation system. Although we did not get a obvious phenotype-specific transgenic plant, there are the same expression levels in target genes of transgenic rice plants compared with the disease and healthy plants which were detected by real-time PCR. The result showes that these vsiRNAs really can lead to the relative expression of target genes down-regulation in Rice.In addition, we have gotten many To transgenic rice seeds which provides a good material for the next experiment to the further research and we also can screen out the rice strain with resistance to RGSV or RRSV.