| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 96 === The soybean seed storage protein β-conglycinin is comprised of three different subunits alpha, alpha' and beta. Genes encoding the three subunits of β-conglycinin have been reported to express only in seeds during mid to late stages in embryogeny. DNA sequences between -257 and-77 bp of the alpha' subunit gene had been reported to function as a seed-specific transcriptional enhancer. This study aims to characterize the promoters of genes encoding the alpha, alpha' and beta subunits of beta-conglycinin in Kaohsiung 10 soybean. A series of constructs containing different promoters fused to beta-glucuronidase (GUS) reporter gene were generated and introduced into soybean protoplasts using polyethylene glycol-directed DNA delivery for transient GUS assays. Our quantitative analyses showed that the alpha' subunit promoter has the highest activity among the three promoters. A 2-fold enhancement was found when one or two copies of the alpha' enhancer were placed in either orientation in front of the duplicate CaMV35S promoter. The alpha' enhancer also increased the activity of the beta subunit promoter. On the other hand, the activity of the beta subunit promoter was increased by the application of glutamine or ABA. Sequence analyses indicate that these promoters contain several kinds of seed-specific motifs and six conserved regions. The recombinant constructs were transformed into Arabidopsis for analyzing the temporal and tissue-specific expression of the promoters. The T2 transgenic plants carrying the alpha' subunit promoter show high GUS activity in mature seeds, indicating high seed-specificity. Interestingly, the alpha' subunit promoter contains all the motifs that have been reported to be necessary for seed-specific expression while the alpha subunit promoter that contains less seed-specific motifs shows lower seed specificity. Our results confirmed that the alpha' subunit promoter directs higher gene expression in seeds than alpha and beta promoters.
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