摘要 本論文之原點為小葉浮萍相減式cDNA 基因庫中受過量銅誘導 的基因-色胺酸合成酵素β鏈，色胺酸合成酵素β鏈是植物體合成色 胺酸過程中最後一個關鍵酵素，色胺酸在植物細胞中是唯一會轉化為 可吸收光能之物質的胺基酸，其下游的衍生物（tryptophan derivatives） 中較為熟知的有葉綠素（chlorophyll）、生長激素（auxin, IAA）、血清 素（serotonin）以及褪黑激素（melatonin）。其中，生長激素IAA 是 具有多功能的植物賀爾蒙，在細胞中作為訊息傳遞者或是基因的調節 者；褪黑激素則是首先發現於人類松果體之神經傳導物質，與光週期 有極高的相關性，並且具有清除自由基之能力。在植物界中，目前熟 知褪黑激素為種子中抵禦過氧化傷害之重要物質。 本論文利用5’端快速基因放大技術（5’-RACE）取得色胺酸合成 酵素β鏈之cDNA 全長，進一步取得基因上游之啟動子，以利日後分 析基因調節之機制，另外由南方氏點墨法判斷出色胺酸合成酵素β鏈 基因在浮萍基因組中應屬於一個中小型的基因家族（gene family）。 本論文同時使用高效液相層析儀追蹤浮萍在過量銅環境中，於不 同時間點下色胺酸與其重要衍生物之含量變化，證實色胺酸合成酵素 β鏈之基因表現與色胺酸及其重要衍生物之產生具有正相關性外，高 效液相層析儀同步自由基清除者偵測系統（HPLC on-line free radical scavenger detection system）更有助於釐清此類衍生物在小葉浮萍中之 生理功能。 Abstract Our previous study has identified several copper responsive genes in duckweed, Lemna minor. One of the genes encodes tryptophan synthase β-chain (TSB) which catalyzes the last step of tryptophan synthesis pathway in plants. The strong expression of TSB under excess of copper may indicate that the elevation of tryptophan synthesis and its subsequent metabolic intermediates could respond to the toxicity imposed by this metal. Tryptophan is a precursor of the phyto-hormone, auxin (IAA) which mediates multiple signals in plants for development, gravitropism and stress responses. Several tryptophan derivatives are also intrinsic light-adsorbing compounds such as chlorophyll, serotonin and melatonin. Melatonin was first discovered in pineal grain of the vertebrates that governs the diurnal and photo-periodic response. Subsequently, it was identified as a free radical scavenger in other organisms such as bacteria and plants. This thesis correlated the timing of gene expression of TSB and the accumulation profiles of the tryptophan and its derived compounds such as IAA and melatonin in order to unfold the physiological response to excess of copper. Genomic Southern analysis showed TSB is a small- to mid-sized gene family. The full-length cDNA was obtained by 5’-RACE and its upstream region was isolated using genomic walking. Time course analysis for tryptophan and its derivatives IAA and melatonin by HPLC showed reasonable correlation between gene expression of TSB and the accumulation of the metabolites. The further application by HPLC on-line free radical scavenger detection system can assist the functional assays for such metabolites.
