Screening Of Soybean GmRCD1 Interaction Protein And Interaction With GmNAC82

RCD1(radical-induced cell death1)protein belongs to the plant SRO(similar to rcd one)protein family.The SRO protein family is involved in the normal growth and development process of plants,and is also an important member of many plant stress-tolerant signaling pathways.In current,it is still unclear that which kind of ligand protein interacts with soybean GmRCD1 and which conserved domains mediate the interaction between RCD1 and ligand proteins.Transcription factors play an important role in the regulation of stress resistance and regulation of functional gene expression.The transcription factor NAC is a plant-specific antiretroviral transcription factor with a conserved NAC domain at the N-terminus and a disordered transcriptional regulatory region at the C-terminus.The role of the transcription factor NAC disorder sequence region in plant stress response pathways is unclear.Therefore,it is highly important to study which kind of transcription factors NAC interacts with GmRCD1 and which conserved domains mediate the interaction.Moreover,it is highly significant in both theory and practice to analyze how transcription factor NAC uses its disordered sequence region to interact with GmRCD1 to regulate plant stress resistance.In this thesis,the GmRCD1 gene was first cloned from soybean cDNA,and the selfactivation of constructed recombinant plasmid was detected.Seventeen candidate interaction proteins that relate to the stress resistance of GmRCD1 were first screened by yeast two-hybrid system,then six selected candidate interaction proteins were further verified the interaction with GmRCD1.The GmNAC82 in soybean is similar to the ANAC82 in Arabidopsis,which has been proved to regulate the stress resistance in Arabidopsis.Therefore,we chose the candidate interaction protein GmNAC82 for further research.GmRCD1 and GmNAC82 proteins were expressed by prokaryotic system,and GST Pull-down experiments showed that they could interact directly in vitro.Bimolecular fluorescence complementation(BiFC)and subcellular localization experiments demonstrated that GmRCD1 interacts with GmNAC82 in plant cell nucleus.To identify the interaction site between GmNAC82 and GmRCD1,we performed a bioinformatics analysis on GmNAC82.Based on the result of Y2 H and sequence characteristics of GmNAC82 disorder sequence region,GmNAC82 with the missing of different gene fragment were cloned by primer design.Simultaneously,the clones of different conversed domains in GmRCD1 were obtained by the primer design based on the character of GmRCD1.Through yeast hybridization experiments,it was confirmed that GmNAC82 interacts with the conserved domain RST in GmRCD1 by its 288-323 motif in disordered region.In summary,the interaction proteins that interact with GmRCD1 were screened by yeast two-hybrid system,providing candidate genes for molecular breeding of soybean;it is the first time revealed that GmRCD1 in soybean interacts with transcription factor GmNAC82,and the interaction between GmRCD1 and GmNAC82 was verified by GST Pull-down and bimolecular fluorescence complementation experiment,which provided a theoretical foundation for the identification of soybean stress resistance pathway;the interaction site between GmRCD1 and GmNAC82 was identified,and the interaction between the disorder sequence domain of transcription factor NAC and the ligand protein was studied,which helps the further understanding of the mechanism in transcription factor NAC mediated plant stress resistance.