Screening And Identification Of The DNA-binding Domain Of The Arabidopsis NAC53 Transcription Factor

The NAC(NAM,ATAF and CUC)family is one of the plant-specific transcription factor families,whose members are widely involved in plant growth,development and stress response.Studies have found that NAC transcription factors bind to target gene promoters and activate or inhibit downstream gene expression to participate in plant defense responses.The research group previously found that the NAC53 transcription factor binds the CCT1 promoter in the Arabidopsis c DNA library screening.This study focuses on the binding of NAC53 to the CCT1 promoter,and the results are as follows:1)The transcription factor NAC53 binds to the CCT1 promoter.Yeast one-hybrid shows that the transcription factor NAC53 binds to the CCT1 promoter,indicating that there is no false positive for the binding relationship.In order to detect the binding relationship in vitro,the expression was induced under the optimal induction conditions(16℃,0.5mmol/L inducer Isopropyl β-D-thiogalactoside)and the purified protein GST-NAC53 was obtained,and the CCT1 promoter was used to synthesize the probe,EMSA It was shown that NAC53 can bind the CCT1 promoter.2)NAC53 mutants with deletion of 4 different domains were obtained.Based on the online protein prediction tools Inter Pro Scan,Uniprot and Jpred,the domain was predicted,and the NAC53 protein has domains at 9-159 aa and 108-165 aa.The comprehensive prediction results named 1-107 aa as A,108-165 aa as B,and 166-549 aa as C.Deletion of different domains resulted in 4 NAC53 mutants: NAC53-AB,NAC53-BC,NAC53-AC,NAC53-B.3)NAC53 N-terminal 1-165 aa binds to the CCT1 promoter.The mutant was induced to express and purified protein was obtained.EMSA showed that the mutant NAC53-AB combined with the CCT1 promoter,and the mutant NAC53-BC,NAC53-AC and NAC53-B did not combine with the CCT1 promoter.Yeast one-hybrid experiments were performed on NAC53 mutants,showing that mutant NAC53-AB binds to the CCT1 promoter,and mutants NAC53-BC,NAC53-AC,NAC53-B do not bind to the CCT1 promoter,indicating that NAC53 N-terminal 1-165 aa is a key region for binding to the CCT1 promoter.4)NAC53 N-terminal 1-165 aa activates CCT1 promoter activity.Dual-luciferase experiments show that NAC53 N-terminal 1-165 aa activates the CCT1 promoter in Arabidopsis protoplasts.5)NAC53 and CCT1 are involved in the resistance of Arabidopsis to Sclerotinia sclerotiorum.After inoculation with Sclerotinia sclerotiorum,the lesion diameter of wild-type plants was about 7.4 mm,while that of nac53-4 plants was about 2.1 mm,and that of cct1 plants was about 4.1 mm.These results indicate that NAC53 and CCT1 negatively regulate the resistance of Arabidopsis to Sclerotinia sclerotiorum.In conclusion,this study found that NAC53 can bind to the CCT1 promoter,the binding domain is located at the N-terminal 1-165 aa,and this region activates the CCT1 promoter activity,and NAC53 and CCT1 are involved in the resistance of Arabidopsis to Sclerotinia sclerotiorum.The results are helpful to reveal the regulatory mechanism and signal transduction of NAC transcription factor in the process of stress response.