文心蘭類Lipid Acyl Hydrolase在阿拉伯芥中的表現與分析

Abstract

文心蘭為目前台灣切花內外銷最大宗之蘭科，是台灣目前頗具發展內外銷潛力之新興花卉，並以日本為主要市場，然而運送的時間過久，嚴重地影響到切花品質，因此如何抑制文心蘭切花老化的研究已成台灣切花出口目前的重要課題。OSAG78進行RT-PCR檢測證實為老化誘導基因，並且在各個組織都有表現，經由NCBI blast 顯示OSAG78可能是文心蘭的一種lipid acyl hydrolase。由RACE (Rapid Amplification of cDNA Ends) 得到完整的OSAG78的cDNA序列，分別用兩種啟動子 (promoter) 35S及AGL5 啟動子將OSAG78表現於阿拉伯芥 (Arabidopsis)，觀察轉殖株之外表型的變化，期望能證明OSAG78與老化的相關性。觀察轉殖阿拉伯芥的外表型後發現，在OSAG78全株表現的狀況下，可以測得較高acyl hydrolase的活性，並且產生植株矮小、花和葉子較小且呈圓形、表皮細胞形狀凹陷較少及表面積較小，還有延緩開花和延遲老化等等的外表型。在花專一表現OSAG78的狀況下，只有在花才會有特異的外表型。推測產生上列外表型的原因可能是acyl hydrolase的活性提高，造成細胞膜磷脂質 (phospholipids) 的過度分解，細胞膜的表面積因而減少，所以細胞形狀改變，進而造成整個植株在外型上的變化。在延緩開花的部份，利用Real-time PCR進行調控開花相關基因的檢測，結果發現延緩開花可能和GA的調控路徑有關，在處理GA之後也發現延緩開花的現象有回復的狀況。在延緩老化的部份，為排除開花授粉後可能造成葉子老化的因素，剪下葉子進行暗處理，確認OSAG78的確延緩葉子老化，推測可能lysophospholipids 大量產生而抑制老化。目前認為OSAG78為老化誘導基因，因過度表現時會延緩老化，推測OSAG78可能有助於抵抗老化逆境。未來可以進行過度表現OSAG78的文心蘭轉殖，期望能得到開花時間較長或是不同花型的文心蘭。

In Taiwan, Oncidium Gower Ramsey is the most potential cutting flower of Orchid exported to Japan at present. However, the transportation takes too much time and seriously damaged the qualities of those cutting flowers. Therefore, how to repress the senescence of the cutting flower of Oncidium has become an essential topic of study. OSAG78 is a senescence-induced gene and expressed throughout the whole plant, and OSAG78, perhaps, is a lipid acyl hydrolase based on the NCBI database. We isolated the full-length cDNA of OSAG78 by using RACE (Rapid Amplification of cDNA Ends), and the ectopic expressions of OSAG78, driven by AGL5 promoter and 35S promoter respectively, in Arabidopsis were analyzed. Compared with wild-type plant, Arabidopsis transformed with 35S::OSAG78 displayed higher lipid acyl hydrolase activity, and also it showed the more circular and smaller flowers and leaves, the smaller surface area of cells, the smoother cell shape, and the relatively smaller plants. Moreover, the 35S::OSAG78 Arabidopsis delayed flowering and postponed senescence of leaves and flowers. However, the AGL5::OSAG78 Arabidopsis displayed the same specific phenotypes in flowers only. These results suggest that these specific phenotypes result from the rising of the acyl hydrolase activity that excessively decomposed the phospholipids in the cell membrane. Moreover, the postponement of flowering in transgenic Arabidopsis can be restores by the treatment of gibberelin (GA), indicating that overexpression of OSAG78 in plant may interfere in the regulation of GA which regulate flowering. Conclusively, OSAG78 shows acyl hydrolase activity, and the overexpression of OSAG78 in plant may produce lysophospholipid which inhibits the function of phospholipid D and then delays the senescence of leaves and postpones the time of flowering. In the future, we hope to overexpress OSAG78 in Oncidium to delay the senescence of Oncidium cutting flower.