Molecular Biology Of RNA1-3 Of Rice Grassy Stunt Virus

Rice grassy stunt virus (RGSV) is a member of Tenuivirus. The filamentous particles of RGSV are ribonucleo-proteins (RNPs), which are composed of a single nucleocapsid (NC) protein and genomic ssRNA segment. It has caused great losses to rice yield in South and Southeast Asia during the 1970's. It also occurred in the south provinces of China, such as Fujian, Taiwan, Guangdong, Guangxi and Hainan province.Compared with the other members of Tenuivirus, studies on RGSV is lagged. The whole sequence of RGSV-IR genome has been determined until 1998 by Toriyama et al. It revaled that RGSV has six genomic RNA segments, RNA segments 1,2,5 and 6 of RGSV corresponds to RNA 1-4 of the other tenuiviurses.And all six RNA segments had an ambisense coding strategy, it is unique not only inTenuivirus, but also in other plant virus. On the basis of the results, we proceeded to study the molecular biology of RNA 1-3 of RGSV-SX.With primers designed according to sequence of RGSV-IR isolate, the cDNA clones covering the full-length of RNA 1-3 of SX isolate were obtained by RT-PCR. We determined the complete nucleotide (nt) sequence of RNAs l(9764nt), 2(4071nt), 3(3120nt) of a Chinese shaxian isolate of rice grassy stunt virus (RGSV-SX) and compared with those of two RGSV isolate (IR and SQfrom Philippines. Sequence analysis showed that the overall nucleotide sequence homology was 99.6% with RNA1, 97.6%, 99.3% with RNA2,98.7%, 91.0% with RNA3, compared with the IR, SC isolates that have been published. RNA2 is most diverged among six RGSV segments, and the nucleotide variation is most in the intergenic region. The results show that it is more homologous between SX and IR than SX and SC. Based on the mentioned results, we conferred that the SX isolate was probably developed from the IR isolate in the Philippines and it is maybe natural occrurrence of reassortment during the process of epidemiology.Using recombinant plasmids containing the vRNA3 NS3 gene and vector of pGEX-2T, we constructed the prokaryotic expression plasmids pGTNS3 which produced the 49K fusion protein of GST-NS3 in E. coll and prepared theantiserum against the fusion protein. So it is possible to provide methods and bases for the further inquiry into the functions of the vRNA3 NS3 gene and its encoded protein.After the system of protoplast is established, we transferred the puried virus particles of RGSV to rice protoplast by the method of PLO. The express of RGSV in rice protoplast is investigated using ELISA and Western-blot. In this study, we sampled according to different time after inoculation.The plant expression vector pCBTNSvS containing the vRNAS NS3 gene wasoobtained by replacing the exterior segment of pCBTNSv6 which constructed bydoctor Zhang Chunmei of our insititute. The recombinant plasmid of pCBTNSvS was introduced into Agrobacterium tumefaciens by triparental mating. The positive clones were then used in the transformation of rice. The transgenic system was established through the exploration of the factors affecting the Agrobacterium tumefaciens transformation and the transgenic rice plants were regenerated. PCR and Southern blotting analysis of the total DNA extracted from transgenic plants showed that the NS3 gene of RGSV had been integrated into the rice genome.