The Research On The Function Of Rice Gene OsAAA1 And Screening Of Its Interaction Proteins

The AAA-ATPase gene family is a large,versatile family whose encoded proteins belong to the AAA~+protein superfamily of P-loop type NTPases.The AAA-ATPase genes are widespread in all organisms and are associated with a variety of cellular activities,including DNA replication,protein degradation,membrane fusion,microtubule disruption,peroxisomal biosynthesis,signal transduction,and gene expression regulation.In recent years,the research on AAA-ATPase gene family in plant disease resistance has been more and more in-depth.OsAAA1 is a new member of the AAA-ATPase family that has been screened by gene chip technology.OsAAA1 is involved in the resistance of rice blast and is up-regulated when induced by Magnaporthe grisea.In this study,molecular identification and resistance analysis of OsAAA1 RNAi transgenic plants as well as its PR1b inducible promoter transgenic plants were carried out.At the same time,the yeast two-hybrid technique was used to screen the interaction proteins of OsAAA1,and we expected to identify the interaction proteins by BiFC.This study laid the foundation for further study on the disease resistance and molecular mechanism of OsAAA1.The main contents are as follows:(1)The molecular identification of the PR1b inducible promoter transgenic plants of OsAAA1 was carried out,and the positive plants were inoculated by M.grisea to perform the expression analysis.The results showed that the inducible promoter PR1b was successfully induced by M.grisea and the expression of OsAAA1gene was activated.The expression level of OsAAA1 was significantly up-regulated after inoculation.The resistance analysis showed that the resistance of PR1b inducible promoter transgenic plants was significantly stronger than the control group,and the lesion size and the amount of M.grisea in leaves were lower than those in control group.(2)OsAAA1 RNAi transgenic plants were identified by PCR and qPCR.The inoculation of M.grisea were carried out on the lines in which OsAAA1 expression was significantly down-regulated.The results showed that RNAi transgenic plants were more susceptible and the leaves even showed acute wilting.The detection of M.grisea content in leaves also showed that RNAi transgenic plants were more susceptible than that of the control plants.(3)Five interaction proteins of OsAAA1 were screened by yeast two-hybrid technique:WRKY19,WRKY66,WRKY68,Pib and Pit.The BiFC vectors of WRKY68 and Pit as well as the N-terminal and C-terminal of OsAAA1 have been successfully constructed.The BiFC assay preliminary confirmed the interaction between OsAAA1N and WRKY68.At the same time,we successfully constructed the bait vector of OsAAA1 for screening a library,which laid the foundation for further screening of its interaction proteins.