離層酸為調控植物生長發育以及抗逆境的重要賀爾蒙，部份與抗逆境有關的基因受離層酸(abscisic acid, ABA)誘導表現，藉由轉錄調控因子(transcription factor)結合至啟動子上的ABA-responsive element (ABRE)轉錄調控序列(cis-element)，誘導抗逆境基因表現。阿拉伯芥基因22 kDa peroxisomal membrane protein-like (PMP22L)具有2個ABREs並受離層酸及逆境誘導表現，其啟動子能持續活化β-glucuronidase (GUS)在成熟的花藥表現，並且受物理性傷害誘導在傷口周圍表現GUS，而10天大的轉殖株在離層酸作用之下，只在根延長區可以偵測到GUS表現，顯示PMP22L的表現具有組織特異性。因此本實驗以刪除啟動子序列的方式，探討PMP22L啟動子各區域調控基因表現的特性。首先由PMP22L轉譯起始密碼上游-1,086 bp到下游+2的啟動子序列，與植物cis-element資料庫(PLACE)進行比對，發現此序列含有多個與逆境有關的cis-elements: ABREs, activation sequence-1 (as-1), dehydration-responsive element (DRE), MYC或MYB factor recognition site以及W box。利用限制酶切割或PCR擴增方式，由5'端或3'端刪除序列，構築出不同長度的啟動子建構序列，分別接上GUS基因表現載體pBI101，並轉殖至阿拉伯芥。以離層酸處理轉殖株，發現缺失兩個ABREs (-630/-586)的轉殖株全株，皆不受離層酸誘導GUS表現，顯示ABREs是PMP22L受離層酸誘導表現所必須的。另外發現，-574/-565區域具有抑制基因於胚軸、根冠和莖頂分生組織表現的功能，推測含有ABREs 和as-1之-630/-565區域，為調控根部特異性表現所必須。接著對轉殖株的葉片進行物理性傷害觀察GUS表現的位置，發現含有W box的-801/-631區域是受物理性傷害誘導所必須的序列。最後觀察各轉殖株的花藥GUS表現，發現-1086/-888以及-801/-567區域，為花藥表現GUS所必須。綜合以上的結果，初步推測PMP22L啟動子含有多個正向或負向調控基因表現的序列，其中的-801/-567區域，調控基因於離層酸或物理性傷害誘導之下表現具有組織特異性，並且調控花藥表現時期的特異性。

　Phytohormone abscisic acid (ABA) regulates stress-responsive gene expression which is mediated by cis-elements named ABREs in their promoter regions. The 22 kDa peroxisomal membrane protein-like (PMP22L) gene promoter has two ABREs and responds to ABA and environmental stresses. GUS histochemical staining also indicated that PMP22L expression in the anthers of opened flowers and rapidly induced by wounding stress. In addition, the expression of PMP22L was also only detected in the roots of 10-day-old seedlings after ABA treatment. In this study, we functionally characterized the promoter region of PMP22L gene to understand regulation machinery of the PMP22L expression by transgenic plants using 5'- or 3'-deletion constructs fused to GUS reporter gene. Firstly, the PMP22L 1.1-kb DNA fragment of the putative promoter region was analysed by PLACE database and several stress-responsive elements: ABREs, activation sequence-1 (as-1), dehydration-responsive element (DRE), MYC or MYB factor recognition site and W box were found. Secondly, various promoter regions containing different stress-responsive elements were constructed. GUS activity was detected in the roots of transgenic plants transformed with full-lengh (-1,086/+2) and various 5'-deletion constructs after ABA treatment, except for the transgenic plants without the ABREs (-630/-586) in the promoter. This result indicated that ABRE was essential for ABA- induced PMP22L expression. The -574/-565 region may contain negative element required for inhibiting GUS activity in the root cap, hypocotyl and shoot apical meristem (SAM). These data showed that the -630/-565 region containing ABREs and as-1 was necessary for the root-specific expression. In addition, the -801/-631 region possessing W box was required for the wounding-induced gene expression. Two upstream regions, -1086/-888 and -801/-567, were necessary for gene expression in anthers. In conclusion, PMP22L expression was regulated by the combination of positive and negative regulatory elements in the promoter. The -801/-567 region may be a minimal ABA- or wounding-responsive unit and necessary for GUS activity in anthers.

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