甘藷Ipomoelin啟動子功能性分析

Abstract

Ipomoelin (IPO) 是一個甘藷(Ipomoea batatas cv. Tainung 57)傷害誘導的防禦蛋白，IPO可以被機械性傷害、茉莉酸甲酯(Methyl Jasmonate)以及乙烯等多種植物逆境與訊息分子所誘導表現，所以是一個可以研究植物如何開啟防禦機制的良好材料。針對實驗室李雨霖學長所釣取的甘藷IPO 啟動子序列全長片段A (位置-1240∼-1)，以及其所建構的啟動子缺失片段B (-1136∼-1 )、 C (-836∼-1 )、D (-496∼-1)、和E (-239∼-1)，進行菸草原生質體暫時性表現，並且進一步建構缺失順向調控區的啟動子長度1232 bp (片段F)，片段F缺失 -1136 bp∼-836 bp之間順向調控序列(cis-acting element)的ethylene response element (ERE, -1107∼-1100）；又建構長度為1235 bp的G片段，缺失 -496 bp∼ -239 bp之間的TGACG-motif (-404~-400)。針對全長啟動子和啟動子缺失片段B、C、D和E，以及含有缺失順向調控序列的片段F與 G，在接上b-glucunonidase(GUS) gene後，分別進行菸草轉殖，分析IPO 啟動子受傷害誘導之機制。在菸草原生質體暫時性表現以及轉殖菸草的實驗中，皆可證實IPO的啟動子是可被茉莉酸甲酯和乙烯所誘導表現的，同時發現茉莉酸甲酯誘導IPO的啟動子表現的倍率比乙烯來得高，感受的時間也較為迅速。而比較不同片段的缺失序列更可以證實：IPO的啟動子感受乙烯誘導的區域為位於-1107 bp 與-1100 bp 之間的ERE；IPO的啟動子感受茉莉酸甲酯誘導的區域為位於-404 bp∼-400 bp 之間的TGACG-motif。且TGACG-motif具有雙重角色：在平時，不受藥劑誘導的情況下，是作為一負向調控者，抑制IPO啟動子，使IPO啟動子在正常的情況下不表現，受傷害才表現；而在處理茉莉酸甲酯的情況之下，TGACG-motif則作為IPO啟動子感受茉莉酸甲酯的調控區域。

Ipomoelin (IPO), a wound-inducible defense protein from sweet potato (Ipomoea batatas cv. Tainung 57), can be induced by wounding, Methyl Jasmonate (MeJA), and ethylene, and is an excellent material to study plant defense system. The full length of IPO promoter, fragment A (position -1240~-1), and its serial deletion fragments B (-1136~-1 ), C (-836~-1 )、D (-496~-1 ), and E (-239~-1 ) were isolated and constructed by Yu-Lin Li, and their tansient expression in tobacco protoplast has been proceeded. Furthermore, motif deletion fragment F, which has deletion of a ethylene response element (ERE, -1107~-1100 ) between fragments B and C and motif deletion fragment G, of which the length is 1235 bp and has deletion of TGACG motif between fragments D and E (-496~-239), were also analyzed. With attachment to the b-glucunonidase(GUS) gene, the full length, serial deletion, and motif deletion promoters were transformed into tobacco, and their expressions stimulated by ethylene and MeJA were studied in the stable transformants. Both the experiments of tobacco transient expression and stable transformation proved the effects of both MeJa and ethylene upon the induction of IPO promoter. Also, ERE that is located between -1107~-1100 was responsible for the induction of ethylene, and TGACG motif positioned -496~-239 was involved in MeJA stimulation in IPO promoter. Also, the induction magnification of MeJA upon IPO promoter was higher than that of ethylene, and the induction response of MeJA to IPO promoter was faster than that of ethylene. Interestingly, TGACG-motif functioned as a negative cis-element repressing the activity of IPO promoter in the absence of MeJA, but upon the addition of MeJA TGACG-motif positively helped IPO promoter expression.