| S-Like RNase is a member of the RNase T2family in plants. It is not only involved in phosphate recycling during senescence and abscission but also involved in plant defense. They can resist on diverse pathogens invitation such as fungi, bacteria and virus. Rice black-streaked dwarf virus (RBSDV), as a member of the genus Fijivirus in the family Reoviridae, infects a number of cereal crop including rice, maize, wheat. The diseases caused by RBSDV lead to severe economic losses of crops.Previous proteomic analysis in our laboratory showed that OsRNase3was induced after RBSDV infection. It reported that OsRNase3could interact with RBSDV P5b in. However, the function of OsRNase3is large unknown. The role of OsRNase3in process of plant and RBSDV interaction need to be further studied. In this study, we did three aspects work to reveal the function of OsRNase3.1. Analysis the expression of OsRNase3in host plants under RBSDV infection.In order to define the effect of RBSDV infection on expression of OsRNase3, real-time fluorescent quantitative PCR was used to calculate the mRNA expression changes of ribonuclease genes. Meanwhile, the expressions of ribonuclease in other two hosts (wheat and maize) were also analyzed. In addition, the expression changes of OsRNase3were compared between susceptible and resistant varieties under RBSDV infection.The results of Real-time PCR showed that RBSDV infection induced the expression of ribonucleases in plants. After RBSDV infection, in Oryza sativa L. japonica. cv. Nipponbar e, the mRNA expression level of OsRNase3in leaf and stem were3.08and6.09folds than the control, in Oryza sativa L. japonica. cv. Huaidao5, the mRNA expression level of OsRNase3in leaf and stem were3.75and4.89folds than the control. According to phyletic evolution analysis, in maize and wheat, ZmaRNS1and TaeRNS4had the most closeast relationship with OsRNase3. Under RBSDV infection, the expression of ZmaRNS1and TaeRNS4were3.49and1.89folds than the control, respectively. Our data indicated that plant ribonucleases might play an important role to defense against virus infection.To analyze whether OsRNase3expression is relate to the accumulation of RBSDV, we analyzed the relationship between the virus accumulation in different organs and the OsRNase3expression levels in rice. The results showed that in Oryza sativa L. japonica. cv. Nipponbare and Huaidao5infected with RBSDV, the expression level of RBSDV P10in stem were5and13.1folds than those in leaf, and the expression of OsRNase3were2.3and4.7folds in stem than in leaf. These data indicated that the accumulation of RBSDV in stem was more than in leaf, there is a correlation between the expression of OsRNase3and virus accumulation. The result further confirmed that OsRNase3played an important role to defense against virus infection.In order to define whether OsRNase3is associated with rice resistance to RBSDV, the expression changes of OsRNase3were studied in resistant and susceptible varieties under RBSDV infection. The results showed that, after RBSDV infection, the expression of OsRNase3in leaf, stem and sheath were4.6,5.1and8.6folds than control in Nipponbare, respectively. After RBSDV infection, the expression of OsRNase3in leaf, stem and sheath were5.2,6.1and9.6folds than control in jindao263. The expression of OsRNase3is higher in resistant than in susceptible varieties, suggesting that OsRNase3was involved in the process of anti-virus.2. Construction of over-expressing and RNAi vector to study the function of OsRNase3In order to further study the function of OsRNase3, we construct over-expressed vector1301-OsRNase3and1300-OsRNase3for benthamiana and rice transformation.500bp gene fragment of OsRNase3was cloned by PCR, and was ligated into pBluescript-intron vector in forward and reverse directions, then inserted into binary expression vector pCAMBIA1300to obtain the RNAi vector1300-OsRNase3-in-OsRNase3.To define the sub-localization in plant cell,1301-GFP-OsRNase3was constructed to infiltrate Nicotiana benthamiana.3. Analyze the interaction between RBSDV proteins and OsRNase3There are two starting locations in RBSDV segment S5ORF2. These two starting locations cause the encoded proteins in a28amine acid difference in length. To clear whether the starting code was related with the interaction between OsRNase3and P5-2, the interaction between OsRNase3and P5-2Δ was analyzed. Yeast two-hybrid result showed that OsRNase3could interacte with both P5-2and P5-2Δ.In order to analyze whether OsRNase3was interact with other proteins of RBSDV, the interaction between OsRNase3and P7-1、P8、P9-1and P10were anlayzed. The results showed that OsRNase3could not interacte with P7-1, P8, P9-1and P10. OsRNase3may interact with P5-2specifically. |
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