The Interacting Protein Identification Of Rice Transcription Factor RST1 And Salt Tolerance Analysis

The transcription factors play a key role in rice stress response pathways.A better understanding of these tolerance mechanisms,through genetically modified the relevant gene and promote effective salt-tolerant genes to be fully functional,can be used to breed crops with improved yield performance under salinity stress.For the past few years,gene cloning and function studies have made a great progress of rice transcription factor,approximately more than 30 transcription factors have cloned Correlating with salt tolerance in rice,that play an important role in regulating salt resistance in different development period of rice.It is well documented that transcription factors belonging to the families of DREB,NAC,MYB,MYC,Cys2/His2 zinc finger,bZIP,AP2/ERF and WRKY are relevant in salt stress tolerance.This thesis takes the rice salt-tolerant mutant rstl as the research object from early identification from laboratory,used the yeast two-hybrid technology to screen the interacting proteins of transcription factor RST1 from rice gene library.And I builded the relevant rice transgenic materials to research the basic physical characteristics and salt resistance,provide some references for explore the salt tolerance mechanism of mutant rstl.First,I screened three interacting proteins of transcription factor RST1 after Yeast rotary validation.Then I maked use of the Pull-down and BiFC technique to further determine the positive interaction relationship between RST1 and CBSX3,and the interaction zone is N terminal and transcriptional repression domain of RST1,indicate CBSX3 may influence the transcriptional regulation activity and transcriptional level of RST1,will affect the expression level of the downstream salt response factor to regulate the salt resistance of plant.Subsequently,I selected two gene CBSX3 and Hsp40,builded the transgenic plants to make a preliminary analysis of their salt resistance.Secondly,I analyzed the tissue expression pattern and subccllular localization of gene CBSX3.The experimental results showed that CBSX3 was localized in the nucleus same as RST1,mainly expressed on the rice ground and the highest amount in the leaf.Then I builded the overexpression strain and RNAi strain of CBSX3,and analyzed the salt resistance of the overexpression strain.When I used 140 mM NaC1 to stress the three lines of OE-CBSX3,all had salt resistant phenotype,aboveground and underground part of Na+/K+ ratio were significantly lower than the wild type,indicate CBSX3 may negative regulate RST1.Quantitative analysis showed that OsCBSX3 response to salt and drought same as RST1,the gene expression after salt stress raised the highest amount both at 3 hours.In both three overexpression line,the expression of CBSX3 had been raised,the expression of RST1 had been reduced.It showed that OsCBSX3 can reduce the OsRST1 expression in RNA level,further reveals the possible interaction mechanism of RST1 and CBSX3.In addition,I also analyzed the subcellular localization of gene Hsp40 and salt resistance of the overexpression strain.The experimental results showed that Hsp40 was mainly located in endoplasmic reticulum and ribosome,indicate it may participate in the protein folding and transport process of RST1.Then I builded the overexpression strain of Hsp40,and analyzed the salt resistance of the overexpression strain.When I stressed the three lines of OE-Hsp40 with 120 mM NaCl,all were salt-tolerant than wild type.Quantitative analysis showed that OsHsp40 response to salt and drought in wild type.In both three overexpression line,the expression of Hsp40 had been raised,the expression of RST1 had been reduced.It showed that OsHsp40 can reduce the OsRST1 expression in RNA level.But,the mechanism on protein level for both two proteins interacting with RST1 remains unclear.In conclusion,I taked advantage of molecular biology,cell biology,biochemistry related experimental techniques to screened protein CBSX3 and Hsp40 which interacting with RST1,and analyzed the tissue expression pattern and subcellular localization of the two proteins.Based on the salt resistance of rice transgenic overexpression strain,preliminarily determined the negative regulatory role between CBSX3,Hsp40 and RST1.