Previous studies indicate that natural antisense transcript,cis-NATZmNAC48,acts as a negative regulator for maize drought stress response gene ZmNAC48.To further characterize the function of cis-NATZmNAC48,we used cis-NATZmNAC48 cDNA sequence and ZmNAC48 protein coding sequence to retrieve maize B73 reference genome and obtain the upstream promoter.PlantCARE and New PLACE were used to predict promoter regulatory elements,which revealed that the promoter of cis-NATZmNAC48 and ZmNAC48 contained not only CAAT-box,TATA-box,and other basic elements,but also hormone response elements and transcription factor binding element.Plant expression vectors of GUS fusion with cis-N AT ZmNAC48 and ZmNAC48 promoters were constructed and transgenic Arabidopsis thaliana was obtained by infecting inflorescences.GUS staining analysis showed that Procis-NATZmNAC48:GUS and ProZmNAC48:GUS was expressed in roots,stems,and leaves of Arabidopsis thaliana.After osmotic stress treatment,the relative expression level of GUS gene and GUS enzyme activity of Procis-NATZmNAC48:GUS transgenic Arabidopsis were significantly decreased and increased significantly in ProZmNAC48:GUS transgenic Arabidopsis,which indicating that both cis-NATZmvAC48 and ZmNAC48 promoters responded to osmotic stress.DNA methylation was one of the regulatory events that affected promoter activity.In this study,we found that DNA methylation modification existed in the promoter region of cis-NATZmNAC48.After osmotic stress treatment,the methylation enrichment changed significantly,but the methylation sites with significant changes were not in the cis-regulatory elements.These results laid an important basis for the analysis of cis-NATZmNAC48 regulation.
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