大豆種子儲存蛋白beta-conglycinin表現在種子發育中後期且具有三個次單位alpha，alpha'和beta。alpha'次單位基因上游-257至-77區域已被報導為種子專一性促進子。這個研究的目的是分離並分析高雄10號大豆儲存蛋白beta-conglycinin的alpha，alpha'和beta次單位啟動子的特性。我們將不同次單位啟動子構築於beta-glucuronidase(GUS)基因之前，並藉由PEG導入DNA的方式暫時性轉殖至大豆原生質體，以GUS表現來定量啟動子的活性。定量分析的結果顯示，在三個啟動子裡，alpha'次單位啟動子具有最高的活性。而一或兩重複alpha'次單位促進子不論正反方向性都具有兩倍促進啟動子活性的效果。此促進子也增加beta次單位啟動子的活性。另一方面，外加穀氨酸或ABA會增加beta次單位啟動子活性。序列分析結果顯示這些次單位啟動子具有多種種子專一性的結合區域，且有六個保守區域。這些啟動子的重組質體轉殖至阿拉伯芥，以轉殖株分析組織專一性表現，在第二代轉殖株，alpha'次單位啟動子表現最高的種子專一性。有趣的是，alpha'次單位啟動子具有最多的種子專一性的結合區域，而alpha次單位啟動子含有較少的種子專一性結合區域表現出較低的活性。我們的結果證實，相較於alpha和beta次單位啟動子alpha'次單位啟動子在種子具有最高的活性。

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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