受高溫影響之文心蘭色氨酸合成酶α單元基因表現的分析

Abstract

文心蘭在夏季高溫的環境下，可縮短花芽發育期，但花序之品質卻明顯降低，花軸分叉數和高度都顯著減少。迄今我們對於影響文心蘭花序分叉數和花軸高度發育的機制，以及參與調控之相關基因的種類及功能卻毫無所知。為期了解夏季高溫影響花序及花軸發育的情形，本論文選殖在高溫下影響文心蘭花軸發育之相關基因，做為日後研究之基礎。實驗採用文心蘭切花品種南西（Oncidium Gower Ramsey）三年株齡之植物個體為材，將帶有20~25公分花軸之植株，分別培養在適溫（25/20℃）及高溫（35/30℃）之下，取其尖端2 cm之組織利用DDRT-PCR（differential display reverse transcription PCR）方法，選殖出表現具有差異性之基因298個。經定序比對，選出一個可能對於花軸生長發育基因有影響的基因，發現此基因在適溫下發育的花軸其表現活性均明顯高於高溫處理的花軸，並將其命名為Oncidium tryptophan synthase alpha subunit (OTSA)。色氨酸(tryptophan)是重要的植物荷爾蒙Indole acetic acid (IAA)的前驅物。前人的研究也已發現，在豌豆中IAA能促進GA 3-oxidase及降低GA 2-oxidase的mRNA表現，進而促使植株莖幹的生長得以變長。設計引子利用RT-PCR及5'RACE的方式，構築其cDNA。以阿拉伯芥的Heat Shock Protein 101的啟動子與其構築在一起，觀察其在高溫影響下表現活性的變化模式。由於目前文心蘭轉殖的技術尚未成熟，故將此融合基因，利用農桿菌轉殖的方式轉入阿拉伯芥中，觀察其基因高溫下之表型的改變。本實驗也調查了不同溫度下阿拉伯芥內生性tryptophan synthase alpha subunit（AtTRP3）基因的表現，發現高溫下阿拉伯芥花軸中AtTRP3的表現量明顯減少。這與文心蘭的情形相似。除此之外，也將其轉殖進入阿拉伯芥的trp3-1突變株中，觀察其基因及表型在不同溫度下的改變。

Development of the Inflorescence of Oncidium could be shortened under high temperature in summer, but the qualities in terms of the length and branch number, drop dramatically. We have no idea about the mechanism of branching, and growing of inflorescence and what kinds of genes involved in regulation. For understanding how Oncidium's Inflorescence develop under high temperature, we cloned genes which affect inflorescence of Oncidium under high temperature. We used three-year-old Gower Ramsey plants as material, and cultured them under moderate temperature（28/23℃）and high temperature（35/30℃）. We used DDRT-PCR（differential display reverse transcription PCR）to clone 298 genes from the top 2 cm of inflorescences. We chose one gene which could affects the growth and development of inflorescence. Expression of this gene is higher under optimal temperature than high temperature. We named it as Oncidium Tryptophan Synthase Alpha subunit(OTSA). Tryptophan is the precursor of the plant hormone-indole acetic acid (IAA). IAA can promote the expression of PsGA 3 oxidase and repress the expression of PsGA 2 oxidase in pea, and promote the growth of stem. We used 5’RACE to obtain cDNA of OTSA and construct a vector with a promoter of Heat Shock Protein 101. Because transformation of Oncidium is difficult, we transform this fusion gene to Arabidopsis thaliana by Agrobacteria. We also investigated the expression of endogenous tryptophan synthase alpha subunit gene(AtTRP3) in Arabidopsis thaliana and found AtTRP3 expressed less amount in inflorescence under high temperature than under optimal temperature.