Screening And Functional Studies Of Proteins Interacting With NAC Transcription Factor GmSJN2

Land salinity is one of the most important environmental problems.Glycine max L.Merr.is an important food and economic crop with moderate salt tolerance.To improve the salt tolerance of soybean,the first key step is to discovery and identify the salt tolerance related gene.The previous study showed that the GmSIN2,a NAC transcription factor gene,could improve salt tolerance of soybean.In order to further investigate its metabolism in salt-stress response of soybean,by screening the Two-hybird cDNA library,we found GmRAB7 and GmPRL1 could interact with GmSIN2.At the same time,we proved the interactive relationship between GmSIN2 and GmRAB7 as well as its function involved in the salt tolerance.The results of this thesis are as follow:Firstly,we screened the forward two-hybrid cDNA library and got 37 different functional proteins by NCBI that might interact with the GmSIN2.Then we selected five different proteins to testify interactive relationship by two-hybrid.Surprisingly,we found two proteins GmRAB7 and GmPRLl worked.Two-Hybrid system,BiFC(Bimolecular fluorescence complementation)and Pull-down experiments indicated that GmRAB7 could interact with GmSIN2 in vitro.Secondly,we divided each protein into several different fragments.We found that the fragment of GmSIN2 that contained NAC subdomain could interact with the fragment of GmRAB7 that contained the RAB subdomain.Additionally,we found the GmRAB7 could also interact with other protein,such as GmNTL1,GmSIN1 and GmNTL7,which also contain NAC domain.Since this,the GmRAB7 was more likely to interact with almost NAC transcription factors to regulate salt response of plant.As method mentioned above,we futher testified the interactive relationship between GmSIN2 and GmPRL1.BiFC showed that these two proteins can interact with each other in nucleus.The result of Two-hybrid suggests that the fragment of GmSIN2 that contained that NAC subdomain could strongly interact with the fragment of GmPRL1 that conrained the WD-repeated subdomain.Based on these results,we analyzed the function of GmRAB7 in the process of salt tolerance:Subcellular localization experiment was in progress to find out that the GmRAB7 located in cytoplasm.Under salt treatment,GmRAB7 over-expression Arabidopsis lines showed that their main root length was significantly longer than WT as GmSIN2 over-expression Arabidopsis lines did.At the same time,we found the GmRab7's homologous gene in Arabidopsis was AtRab7 gene.Loss-of-function mutants of AtRab7 displayed shorter root length than WT.This result indirectly stated that the GmRAB7 gene play an important role in salt response.