The Research On The Disease Resisitance Mechanisms Of OsAAA₁ And The Expression Pattern Of Four Unknown Gene OsAAA In Rice

There is AAA-ATPase gene family in all kinds of living organism,and it is a large superfamily of protein.AAA-ATPase family play a role in distinct cellular processes including protein unfolding and degradation,protein disaggregation,microtubule disassembly,membrane fusion and peroxisome biogenesis.In recent years,AAA-ATPase family members have been studied more and more thorough,and involved in regulating plant disease response.In the previous research,it was found that OsAAA₁ and four new OsAAA genes were involved in rice blast resistance response through gene chip.OsAAA₁ was induced express by Magnaporthe oryza and salicylic acid,respectively.The overexpression of OsAAA₁ in rice can confer to rice blast disease and bacterial blight.By yeast two-hybrid,OsAAA₁ was found to interact with WRKY19,WRKY66 and WRKY68 transcription factors,disease-resistant proteins Pib and Pit.Therefore,this study aims at the preliminary research on the disease resistance mechanisms of OsAAA₁ and the expression pattern analysis of four unknown gene OsAAA in rice,which can lay foundation for discovering potential resistance resources and learning rice disease resistance regulation mechanisms,and then cultivated rice resistance varieties are of great significance.The research results are as follows:（1）In order to verify OsAAA₁ interacting proteins screened by yeast two-hybrid,the Bi FC expression vector of WRKY19,WRKY66 and Pib were constructed,and the Bi FC verification experiments have been finished.The results of Bi FC fluorescence observation suggested that the N-terminal of WRKY19 and WRKY66 interact with the N-terminal of OsAAA₁ ,the N-terminal conserved core domain of Pib and Pit interact with the C-terminal of OsAAA₁ ,which were the same with the result of yeast two-hybrid.（2）In order to further verify the protein interaction of OsAAA₁ in rice vivo.The overexpression vector with Flag tagged OsAAA₁ was successfully constructed.By a series of experiments including genetic transformation by agrobacterium-mediated,DNA identification as well as detection of expression,the results showed that ten T₀generation transgenic position plants can express the fusion protein of OsAAA₁ and Flag.It laid a foundation for the subsequent use of Co IP technology to verify the protein interaction of OsAAA₁ in vivo.（3）To explore the regulatory role of OsAAA₁ in SA signaling pathway,after BTH induced,the expression of WRKY45 marked gene in the SA signaling and disease resistance was compared with Nip and OsAAA₁ RNAi transgenic plants.The results showed that the expression of WRKY45 in OsAAA₁ RNAi transgenic plants were lower than in Nip after BTH induced.The results of resistance identification of rice blast showed that after BTH treatment,the RNAi plants of OsAAA₁ were obviously susceptible to rice blast,which indicated that OsAAA₁ blocked SA mediated resistance.（4）In order to analyze the expression patterns of four unkown OsAAA genes involved in rice blast resistance,Nipponbare was used as materials to be stressed by Magnaporthe oryza and treated by the plant hormones BTH,Me JA,ABA,KT,BR and so on,which aimed to analyze the changes in expression levels of these four unkown OsAAA before and after treatment.The results demonstrated that,the expression of Os01g19260 and Os05g51130 were up-regulation,and Os09g26260 and Os12g24320 were down-regulation after Magnaporthe oryza induced.Os01g19260gene was up-regulation after BTH and Me JA application,down-regulation after KT application,no significant changes after ABA and BR application.Me JA induced up-regulation and BR induced down-regulation were confirmed with Os05g51130after treatment,OsO5g51130 were not obviously changes after BTH,ABA and KT application.The expression of Os09g26260 were up-regulation after BTH,Me JA and BR treating Nip,down-regulation after ABA treatment,no significant changes after KT treatment.Os12g24320 gene was up-regulation after BTH and ABA application,possibly up-regulation after Me JA and KT application,no obviously changes after BR application.At present,it is supported that four genes play the role in the disease resistance response,as well as phytohormone.The above results laid the foundation for subsequent research on the function of OsAAA genes.