甘藷葉部胰蛋白用脢抑制因數基因啟動子的選殖與特性之研究

Abstract

一個甘藷葉部受缺水逆境誘導表現的蛋白質經純化後，依據其蛋白質序列比對得知屬於potato inhibitor l family。故命名為甘藷葉部胰蛋白脢抑制因數基因SPLTI(Sweet Potato Leaf Trypsin Inhibitor)。SPLTI在幼葉會恆定表現；只有經缺水處理後才誘導表現於甘藷成熟的葉部。根據SPLTI基因的表現研究，發現除會受低溫與滲透壓逆境誘導外，若以ABA、MeJA等相關賀爾蒙處理亦可被誘導，而且經機械性傷害處理後屬於局部誘導表現。 本論文主要探討SPLTI基因啟動子在阿拉伯芥中的調節表現機制。利用improved PCR-based genome walking方法，進行分子選殖及啟動子調控區位(cis-acting elements sites)分析。選殖出約1.8kb SPLTI基因啟動子DNA片段，其包含兩個ABRE-like、兩個ACGTG-box core element與兩個W-box；七個MYB與八個MYC蛋白結合序列及五個DRE-like。將啟動子片段接上uidA (GUS)-coding sequence建構轉殖載體，進行阿拉伯芥轉殖。籍由植物基因轉殖技術，在阿拉伯芥中得知SPLTI基因啟動子之調控機制主要受乾燥、低溫及傷害誘導表現，其調控機制亦會受ABA、SA與MeJA等賀爾蒙誘導表現。經乾燥、低溫處理誘導之GUS活性表現染色分析，在葉部葉脈最為明顯，其次為葉肉組織。轉殖株經傷害誘導GUS活性表現則屬於局部性表現，而非全株性誘導表現。 更進一步針對SPLTI基因啟動子和乾旱誘導有關的調節序列區位，進行啟動子不同truncated片段阿拉伯芥轉殖株之GUS活性分析。結果顯示-573~-19之起動子DNA片段相較於起動子其他truncated片段，誘導表現出最低的GUS活性；而刪除-573~-445起動子DNA片段，則誘導表現GUS活性則有上升趨勢。這個現象說明-573~-445起動子DNA片段中也許存在負調控因數的結合序列。兩個起動子truncated片段之組合，分別將-1327~-1043與-1065~-890片段與-444~-19片段相接，其阿拉伯芥轉殖株受乾燥處理誘導表現之GUS活性皆有顯著上升。這現象至少說明在-1327~-1043與-1065~-890啟動子DNA片段中存在SPLTI基因啟動子和乾旱誘導有關的正向調節序列區。

One small drought-inducible proteinase inhibitor protein, SPLTI, was isolation from leaves of sweet potato (Ipomoea batatas Lam.) plants that is a member of the potato inhibitor I family. Northern analysis indicated that water-deficit; low temperature and mechanical wounding can induce SPLTI gene expression. To understand the role of promoter region of SPLTI in regulating gene expression, the -1.8 KB 5-flanking region of SPLTI was further cloned and sequenced by PCR-based genomic walking method. A number of putative regulatory motifs were identified including two ABRE-like, two W-box, five DRE-like plus seven MYB and eight MYC protein binding sequences. The promoter of SPLTI was fused to the uidA (GUS)-coding sequence and the resulting construct was used to transform Arabidopsis. The β-glucronidase (GUS) reporter gene driven by the SPLTI promoter was induced by dehydration, low temperature and mechanical wounding treatments. Histochemical analysis of GUS activity revealed that the SPLTI promoter functions in most of the plant tissues during water-deficit and low temperature stresses. Mechanical wounding locally, but not systemically induced the expression of GUS in transgenic plants. Furthermore, the cis-acting elements of SPLTI promoter region by deletion analysis in regulating gene expression showed that the region from -573 to -423 of SPLTI promoter might contain negative regulatory cis-acting elements. Two regions from -1327 to -1044 and -1065 to -787 of SPLTI promoter was required for dehydration-induced activated expression of SPLTI.