The Function Of OsRNS4 In The Process Of Rice Black-streaked Dwarf Virus Infection

Rice black-streaked dwarf virus(RBSDV)can harm rice,corn,wheat and other gramineous crops,and causes the rice black stripe dwarf disease and maize rough shrinkage disease,so that caused serious influence on agricultural production.In the early stage,proteomics studies showed that RBSDV can induce the expression of the protein of the rice ribonuclease.The ribonuclease has highest homology with Os RNS4,but with the logged-in Os RNS4 sequence differences in C side has five amino acids,induced RBSDV ribonuclease here still presses Os RNS4 named.In order to analyze the function of Os RNS4 in the infection process of RBSDV,this study analyzed the influence of RBSDV infection on expression of Os RNS4 in rice different tissues.The interaction between Os RNS4 and RBSDV P5-2 was verified.The transgenic plants of overexpression and the interference of Os RNS4 were constructed,and the role of Os RNS4 in virus infection was clearly determined.The results were as follows:1.The expression of Os RNS4 in different tissues of rice after RBSDV infection was clarified.After artificial inoculated RBSDV for Nip and Kitaake,in 3 leaf stage,we extracted total RNA,reversed transcription,then analyzed by PCR and q RT-PCR.The results of PCR showed that the expression of Os RNS4 was increased after RBSDV infection.The results of q RT-PCR showed that the expression of Os RNS4 had the highest expression in the leaves,followed by the stem,with the lowest expression in the root system in Nip and Kitaake without RBSDV infection.After RBSDV infection,the expression of Os RNS4 induced respectively in roots,stems and leaves in Nip and Kitaake.The induction expression of Os RNS4 in the stem was the highest after RBSDV infection.For analyzing the induced quantity of Os RNS4 whether has positively correlated with accumulation of RBDV,the accumulation of RBSDV in rice different tissue were analyzed,and the results show that the expression of RBSDV in the stem was higher than that in the leaves,but the accumulation in the root was the highest.The results showed that the induced expression of Os RNS4 had no direct correlation with the accumulation of virus.2.The resistance identification of rice variety to RBSDV showed that the resistance of 9311 was significantly higher than Nip.The incidence of 9311 was 10%,while the incidence of Nip was 78%.To analysis whether Os RNS4 associated with the resistance of rice to RBSDV,we analyzed the expression of Os RNS4 change by q RT-PCR after the inoculation of RBSDV Nip and 9311.The results show that after inoculated RBSDV the expression of Os RNS4 was 28.89 times that of unvaccinated controls in 9311,and he expression of Os RNS4 was 1.65 times that of unvaccinated controls in Nip.It is suggested that Os RNS4 may participate in the defense process of rice to RBSDV.3.The overexpression and interference of Os RNS4 transgenic rice were constructed.After screening the Os RNS4 transgenic plants of pure T2 generation,the expression of Os RNS4 in T2 transgenic plants was analyzed by q RT-PCR.The results showed that the expression of Os RNS4 in the overexpression of transgenic strains of OE-T2-3,OE-T1-12,OE-T2-50 and OE-T2-95 were 3.29,2.47,3.15 and 7.90 times respectively than that in the wild type.The expression levels of Os RNS4 in the interference strains were significantly reduced,and the relative expression of Os RNS4 in RNAi-T1-4,RNAi-T2-46 and RNAi-T2-140 lines were 0.48,0.01 and 0.08 times respectively than that in the wild type.4.In order to clarify the role of Os RNS4,the overexpression and interference transgenic rice inoculated RBSDV by artificial inoculation.The results showed that the incidence of RBSDV was decreased after overexpression of Os RNS4,and the increased incidence of RBSDV after the interference of Os RNS4.The accumulation of RBSDV in transgenic plants was analyzed by RT-PCR and q RT-PCR,and the results showed that the expression levels of RBSDV P5-2 and P10 were lower in the overexpression Os RNS4 strain than that in the wild type.This indicated that the amount of virus accumulation is low.The above results indicated that Os RNS4 plays a role in defending against RBSDV.5.The interaction between Os RNS4 and RBSDV p5-2 was analyzed.Studies have shown that Os RNS4 can interoperate with RBSDV P5-2.Because the Os RNS4 sequence obtained in this study is different from the five amino acids in the c-terminal of the logged Os RNS4,the interaction between Os RNS4 and RBSDV P5-2 induced by RBSDV infection was analyzed.And the results showed that Os RNS4 could be directly interacted with RBSDV P5-2 by Y2 H and Bi FC.6.Studies have reported that ABA can enhance the resistance of plants to the virus,while Os RNS4 can enhance the sensitivity of rice to ABA.Previous studies have shown that RBSDV infection can change the expression of key genes in ABA synthesis and metabolism.In order to analyze whether Os RNS4 affects the resistance to the virus through the ABA pathway.We analyzed the interaction between Os RNS4 and ABA synthesis pathway key enzymes Os NCED1,Os NCED2 and Os NCED4 by Y2 H,It was found that Os RNS4 could be directly interacted with Os NCED1 and Os NCED4,and it was speculated that Os RNS4 could affect ABA pathway through interaction with Os NCEDs,thereby affecting the resistance to virus.7.We constructed the RBSDV P5-2 transgenic rice.The expression of Os RNS4 in RBSDV P-2 transgenic rice showed that the expression of Os RNS4 was significantly lower than that of the control group after overexpression of RBSDV P5-2,indicating that RBSDV P5-2 could inhibit the expression of Os RNS4.In addition,the expression of ABA synthesis key genes in RBSDV P5-2 transgenic rice was analyzed.The results showed that the expression of Os NCED2 and Os NCED5 were all significantly decreased,and the results of Os NCED1,Os NCED3 and Os NCED4 were not consistent in different transgenic strains,and further analysis was needed.The above results indicated that overexpressing Os RNS4 enhanced resistance to virus.Speculate that Os RNS4 strengthen the ABA synthesis pathway through with Os NCEDs,and enhance the resistance of rice to the virus.But the virus can through inhibit Os RNS4 expression by the interaction P5-2 and Os RNS4,,which is advantageous to the infection of RBSDV.