Genetically Engineered Rice Varieties Resistant To Rice Stripe Disease By RNAi

Rice stripe virus as the typical member of Tenuivirus causes the rice stripe disease which is the main virus disease with the large distribution, serious infection and harmful influences. In our study, the inverse-repeated sequences of CP and SP were recombinanted into the rice genome in order to gain resistant plants respectively.RNA of Rice stripe virus was extracted from the leaf with obvious disease symptoms of the disease-sensitive cultivar Suyunuo planted in our lab trial fields. CP and SP gene fragments were amplified via RT-PCR amplification. Then these fragments were digested with BglII and XhoI restriction enzymes, and the target fragments were recombinated with intermediate vector pUCCRNAi, respectively, to gain the recombinated vectors pUCCRNAi-CP-1, pUCCRNAi-SP-1 which were digested with BamHI and SalI in order to recombinated with the CP and SP fragments digested with BglII and XhoI. Then RNAi vectors pUCCRNAi-CP-2 and pUCCRNAi-SP-2 were constructed. Through sequencing CP and SP target fragments of the constructed vectors and blasting analysis of the sequencing results with the published virus genome sequence in the NCBI database, we found that these two fragments had 5bp and 2bp difference with the most homologous isolates, respectively. Based on the above vectors, we used PstI to digest these two vectors with suitable restriction enzymes to recombinate with the plant expression vector pCam23A digested with the same enzymes to construct pCam23A- CP and pCam23A-SP, respectively. Trough Agrobacterium- mediated, the inverse-repeated sequences of CP and SP were transformed into the genome of Guanglingxiangjing and Suyunuo cultivars. After screening and differentiation, we gained target transgenic plants.The PCR results indicated that CP and SP genes were integrated into the rice genome. And we took an artificial inoculation test on transgenic plant with the control varieties, japonica Guanglingxiangjing and Suyunuo, which were only transformed with resistance selective gene. The disease-resistant results suggested that the four lines of transgenic plants all acquired the interference ability to restrain the expression of the single endo-virus gene, which caused rice to have the notable improvement in the resistance to Rice stripe virus.The detection, via RT-PCR, of injected plants with CP protein (SP protein) with inverse-repeated structure and the controls showed that among the transgenic plants no SP (CP)gene was detected 10 days after the infection while virus RNA were detected in the controls. It also indicated that the replication of virus RNA was restrained obviously in transgenic plants and the transgenic plants were obtained the interference ability to restrain the expression of the endo-virus gene. Meanwhile, we verified that the replication of the coat protein (CP) and the disease specific protein (SP) in host and the disease infection had close connections, each of which caused no disease separately.