甘藷Ipomoelin啟動子特性分析

Abstract

Ipomoelin (IPO) 是一個甘藷(Ipomoea batatas cv. Tainung 57)中的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，我們用iPCR 以及tail PCR的方法找出自IPO 基因轉譯起點之前的1482個鹽基對的啟動子序列，為了要找出能感受茉莉酸甲酯、乙烯的專一序列，我們將IPO啟動子裁剪成五種長短不同的片段，在這五種片段後接上 β-glucuronidase (GUS)報導基因後構築到表現載體pBI221SI100中，並將這五種不同載體送入煙草原生質體中作暫時性表現，結果顯示在IPO 基因轉譯起始點算起-552 到 -921間的序列會受到茉莉酸甲酯的誘導，而在-1176到 -1438間的序列會受到乙烯誘導，而後針對-552 到 -921間會被茉莉酸甲酯誘導的序列進行缺失片段實驗，以三個新的缺失片段載體進行試驗，結果找到一段CACACGTC片段能專一感受茉莉酸甲酯的誘導，此結果與之後進行的Gel Mobility Shift Assay相同，除此之外Gel Mobility Shift Assay還找到一段能被茉莉酸甲酯誘導核蛋白附著的序列TCTAATTTTA，這個序列與阿拉伯芥中茉莉酸甲酯的誘導序列相似。

Ipomoelin (IPO), a wounding-inducible defense protein from sweet potato, (Ipomoea batatas cv. Tainong 57) can be induced by wounding , Methyl Jasmonate (MJ) and ethylene, and is an excellent target to study plants defense system. The IPO promoter was isolated by iPCR and tail PCR from sweet potato, and contains 1482 bp of the upstream region from the translational start codon of IPO. Five different deletions in the IPO promoter region were created in order to locate its MJ and ethylene-responsive regions. Theβ-glucuronidase (GUS) reporter gene was fused to these promoter segments in the sense direction on PBI221SI100 vector, and the transient expression of these constructs induced by MJ and ethylene in tobacco mesophyll protoplast was studied. Results indicated that the MJ-respond region is from the -552 to -921 bp and the ethylene-respond region is between the -1176 and -1482 bp form the start codon of IPO protein. Also, the regulatory elements of MJ and ethylene in IPO promoter are different.The further 3 new expression constructs each with deletion of one element from the -552 to -921 bp were used to locate the MJ-respond element, the result revealing that the CACACGTC element is one of the responsive element for MJ. The similar result was also confirmed by gel mobility shift assay which point out the involvement of CACACGTC element to MJ response. Besides, the result of gel shift experiment demonstrated the main MJ-responding element is ‘‘TCTAATTTTA,’’ which is similar to the MJ-responding element from Arabidopsis thaliana .