Transformation Of CP And SP Genes Of Rice Stirpe Virus And Sequence Analysis Of RNA4

Rice stripe virus (RSV), a type member of Tenuivirus. It has caused great decreases to rice yields in sixteen provinces in China. The previous studies on the pathology of RSV in our institute provided evidence that CP (coat protein, CP) and SP (disease-specific protein, SP) are possible pathogenicity related protein. So, to further study the function of CP and SP gene, we constructed two plant -expression vectors pCA1300-CP and pCA1300-SP separately containing CP and SP gene. 35S promoter. Nos-ter non-coding sequence and vector pCAMBIA1300. Recombinant Plasmids pCA1300-CP and pCA1300-SP were introduced into Agrobacterium LBA4404 by DNA-directed transformation, electroporation and triparental mating separately. The results showed that the two former methods are better than the last one. Then the transformation was done mediated by Agrobacterium. PCR analysis and PCR-Southern blot hybridization by using the total DNA of regenerative plants showed primarily that CP and SP gene has been transferred into rice respectively. And it also showed that the traget genes in some regenerative plants have been transcripted into mRNA by using PCR-Southern blot hybridization. These results showed primarily that CP . SP haven't pathogenicitic function respectively.To further study the function of SP gene and the evolutional relationshipe of tRSV, RNA4 segment of a severe isolate of RSV-SQ has been cloned and sequenced, and shown to be 2157bp in length. Comparison of the vRNA4 ORF of SQ isolate with PJ isolate of LiaoNing province, T, M of Japan and CX of YunNan province revealed 98.1%, 98.3%, 98.3%, and 96.1% identity at the nucleotide sequence level, 98.4%, 98.1%, 97.8%, and 94.0% in vcRNA4 ORF, 94.0%, 94.8%, 89.4% and 85.4% in RNA4 IR; And 99.4%, 100.0%, 98.9%, 98.9% identity in vRNA4 ORF at the amino acid level, 99.0% in PJ isolate and 97.6% in other three isolates in vcRNA4 ORF. We also find that SQ isolate has 96%. 96%. 95%. 90% sequence identity with PJ. T. M and CX isolates respectively at the nucleotide sequence level of total RNA4, and 96%. 95%. 91%respectively for PJ isolate with T, M, CX isolates, 95%, 91 % for T with M and CX isolates, 92% for M with CX. These results demonstrated the closer phylogenetic relationship between SQ and T,PJ,M isolates than that between SQ and CX isolate although both SQ and CX belong to Chinese isolates. The non-encoding sequences of 5 '-termini among the five isolates were identical and those of 3 '-termini were also identical except for a 2- nucleotide variation in CX isolate. Furthermore, IR sequences were rich in U and A residues where two distant hairpin structures could be formed according to computer-assisted folding analysis. Possible variaton rule was also disscussed.