Study On The Interaction Between Rice Stripe Virus Functional Proteins And Proteins Of Oryza Sativa

Rice stripe disease caused by Rice stripe virus (RSV) is an important disease to rice. And it distributes in a large scale of China, has caused great harm to agricultural production, RSV is an multipartite virus, its gnome consists of four kinds of single-stranded RNA (ssRNA).They encodes seven proteins in all, including RNA-dependent-RNA polymerase(RdRp), NS2, NSvc2, CP, NS3, SP, NSvc4.NB15 and NW1 was chosen in this study. The two proteins were selected respectively from rice cultivars (strains)"Wuyujing 3 natural mutant"and"Wuyujing 3"by yeast two hybrid system using RSV NSvc4 as bait. The two proteins were selected to investigate interactions with CP,SP,and NSvc4. Total RNA was extracted from RSV-infected rice leaves and the full-length ORF of the NB15 and NW1 were amplified by RT-PCR with primer pairs designed according to coresponding cDNA sequences. The specific fragments were cloned into pMD18-T respectively. Recomba- nts containing NB15 ORF and NW1 ORF were digested and fragments were ligated into linearized pGADT7 vector individually. The recombinant plasmids were named as pGAD-NB15 and pGAD-NW1 respectively. In order to detect the interaction of CP, SP and NSvc4 with NB15 and NW1 respectivly, the recombinants were co-transformed into yeast strain AH109 individually, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: NB15 and NW1 interact with NSvc4, but not with CP and SP.Using specific primer pairs of gene NB15 and NW1, the expression of NB15, NW1 mRNA in the health and RSV-infected rice leaves were examined by Real-Time PCR. The difference of mRNA expression were compared, and the effect on mRNA expression was analyzed .The results showed: the expression of NB15 mRNA in the RSV-infected rice leaves was down-regulated compared with that in the health rice leaves; The expression of NW1 mRNA in the RSV-infected rice leaves was up-regulated compared with that in the health rice leaves.The expression vector of NB15 gene was constructed by means of the expression vector pET-29a. The recombinants pET -NB15 was transformed into E.coli BL21(DE3) and induced to express. A 27KDa fusion protein product was detected by SDS-PAGE which showed that the expressed protein is in the same size as expected, suggesting that it was expressed in Escherichia coli BL21 successfully. And then, the pured protein was obtained to immune healthy male rabbit to make the antiserum to NB15.Some days after, the antiserum of NB15 was producted and was detected by ELISA(12800), and was used in the detection of healthy rice and RSV-infected rice by Western-blot. The results of western-blot showed that protein NB15 of rice could react wth antiserum specifically, and the expression of NB15 in healthy rice increased compared with that in the virus infected rice leaves. So, the interaction of NB15 with NSvc4 may have affected the expression of rice protein. And the preparation of antiserum of NB15 can be used in the study of immune colloidal gold and cell location. Besides the reserches above, RSV NS3 gene was amplified by RT-PCR and inserted into bait vector pGBKT7 and prey vector pGADT7 respectively. In order to detect the self-activation of NS3 and toxicity to yeast cell, the recombinants were transformed into yeast strain AH109 individually, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: NS3 did not self-activate and have no toxictiy to yeast cell and can't interact with itself, either. To investigate the interactions of NS3 with SP, CP and NSvc4, the recombinants plasmids designed as NS3-PGAD/SP-PG-BK, NS3-PGBK/SP-PGAD, NS3-PGAD/CP-PGBK, NS3-PGBK/CP-PGAD, NS3-PGAD/NSvc4-PGBK, NS3-PGBK/NSvc4-PGAD were cotransform ed into yeast strain AH109, and the transformants were plated on the selective media. The results showed that: NS3 could interact with SP, but no interactions with CP and NSvc4.