Study On The Interaction Between Functional Protein Of Rice Stripe Virus And Rice Protein

Rice stripe disease caused by Rice stirpe virus (RSV) is an important disease to rice. It distributes in a large scale of China and brings huge loss to people. In order to further study the function of some viral proteins, pathogenic mechanism of the virus, and molecular mechanisms of plant resistance and susceptibility to RSV, we researched the relationship between viral proteins and host proteins at molecular level. Based on library screening, the yeast two hybrid system was employed to further confirm the interaction of viral protein and host protein. we also studied the potential interactions of the function protein NS2 with other proteins encoded by RSV.Using rice libraries ("Wuyujing 3"and"KT95-418"), we previourly identified a number of rice proteins that interact with used RSV CP,SP and NSVc4 respectively in the yeast two hybrid system. In this study, 2 interesting clones namely CB18 and NB17 were selected from the yeast two-hybrid screening results. The full-length gene was amplified by RT-PCR, and were then inserted into yeast two-hybrid prey vector pGADT7, thus constructing the recombinants pGAD-CB18 and pGAD-NB17. After that the recombinants pGAD-CB18 or pGAD-NB17 were co-transformed into yeast cell AH109 with bait plasmids pGBK-CP, pGBK-SP and pGBK-NSvc4 respectively, and the co-transformants were plated on different synthetic dropout nutrient medium. The results showed that CB18 interacted with CP weakly but not with NSvc4 and SP; NB17 could interact with NSvc4 but not CP and SP.The expressions of CB18 and NB17 mRNA in the healthy and RSV-infected rice leaves were examined by Real-Time PCR. The differences of mRNA expression were compared, supposing that any changes in the mRNA expression could be a result of RSV infection or interactions with RSV encoded protein. The results showed: the expression of CB18 mRNA in the RSV-infected rice leaves was up-regulated compared with that in the health rice leaves; The expression of NB17 mRNA in the RSV-infected rice leaves was down-regulated compared with that in the health rice leaves.Fusion protein expression vector PGEX-CB18 was constructed by using prokaryotic expression vector PGEX-4T-1. The recombinants pGEX-CB18 were transformed into E.coli BL21(DE3) and induced to express. The expression products of GST-fusion protein were detected by SDS-PAGE which showed that it is in same size as expected. Then antiserum of CB18 was prepared using the expressed protein, Western-blot analysis showed that the antiserums could react with the expression products of the fusion genes specifically. Then the antiserum was used to investigate the protein expression difference in RSV infection rice and healthy rice. The results showed that: the protein expression of CB18 in the RSV infected rice is more than that in the healthy rice.Additionally, RSV NS2 genes was amplified by RT-PCR and inserted into yeast two-hybrid system bait vector pGBKT7 and prey vector pGADT7 respectively. The recombinants were transformed into yeast cell AH109 respectively, and plated on the different synthetic dropout nutrient medium to detect the self-activity .Then, the recombinants were co-transformed into yeast cell AH109 with yeast expression vector CP, SP and NSvc4 respectively and plated on the different synthetic dropout nutrient medium to detect the interactions between NS2 and CP, SP, and NSVc4. The results showed: NS2 have no self-activity .it could interact with itself, but could not interact with CP, SP, and NSVc4.