Molecular Cloning And Characterization Of The Promoter Of 8S Globulin Gene From Vigna Radiata

According to the mRNA sequences of the 3 subunits of mung-bean seed storage protein 8S globin, we designed 3 pairs of specific reserved primers for each subunit. The putative promoter sequences of the 3 subunits, alpha, alpha'and beta were amplified by the method of genome-walking using mung bean genomic DNA as template. The promoter of alpha subunit was 472bp with the GeneBank accession number FJ792642, while the alpha' promoter was784bp with accession number HQ214071 and the beta promoter was 661bp with accession number GU176353. Bioinformatics analysis was carried iut on these promoter sequences with online promoter prediction tools such as plantCARE, PLACE and softberry. Not only several core elements which are usually present in promoters such as TATA box and CAAT box, but also some seed-specific cis-regulational elements such as RY motif and G box were found in our isolated promoters. To determine the transcriptional activity of the three putative promoters, three expression vectors were constructed by replacing the original 35S promoter in plasmid pBI121-GFP with the isolated alpha, alpha'and beta promoters. Resultant expression vectors were subsequently transformed into the mung bean cotyledon proplasts for transient expression. Reporter GFP expressed highly in cotyledon protoplasts as detected by confocal microscopy, demonstrating the specific activity of 8SG promoters in driving gene expression. This study also provided the promoter as efficient regulatory elements for plant seed bioreactor..