蝴蝶蘭乙烯形成酶cDNA之選殖及演繹蛋白分析

Molecular Cloning and Analysis of the cDNA Encoding Ethylene-Forming Enzyme in Phalaenopsis

乙烯在蝴蝶蘭花瓣老化過程中扮演重要的角色，而乙烯的產生係其生 合成途徑上相關酵素活性增加所致，經由調節這些酵素的基因表現，可影響乙烯的生成量及老化的過程。本研究的目的，是選殖與蝴蝶蘭花瓣老化相關的乙烯形成酶cDNA，研究其構造，並探討此基因在花瓣老化過程中所扮演的角色。為了獲得篩選基因所用的探針（probe），本研究首先利用蛋白質演化保留區（conserved region）設計兩段寡核苷酸為聚合酶連鎖反應（polymerase chain reaction; PCR）的引子（primer），以蝴蝶蘭基因組DNA（genomic DNA）為模版進行反應，得到兩段乙烯形成酶基因片段，命名為414及419。為了選殖蝴蝶蘭乙烯形成酶cDNA，我們由老化花器中，抽取poly（A）⁺RNA構築cDNA庫（cDNA library），以414及419的乙烯形或酶基因片段為探針，篩選廿萬個 溶斑形成單位（plaque-forming unit），獲得997個具有雜交反應的溶斑。其中pPEFEA之選殖系經純化並進行核苷酸定序分析，含有1349bp ，具有326個胺基酸的解碼框架（open reading frame），其演譯之蛋白質分子量為37132Da。將pPEFEA與番茄乙烯形成cDNA（pT0M13）比較，顯示兩者所解碼之胺基酸序列有72.2%的相似性。分析pPEFEA選殖系所演繹之蛋白質結構，具有一個醣基結合位置（N-glycosylation site; 101-NIS-103），於113至142的胺基酸序列，為 一兼親疏水性（amphipathic）的α-螺旋結構，在此區域內尚具有一個可形成leucine zipper的演化保留區。另外，pPEFEA演繹的胺基酸序列也包含了12個以Fe（Ⅱ）及抗壞血酸（ascorbate）為輔因子（cofactor）之酵素演化保留殘基（conserved residue）。以南方氏雜交分析（Southern analysis）pPEFEA在蝴蝶蘭基因組內之結構，顯示其為一單一拷貝基因。以乙烯形成酶cDNA為探針進行北方雜交分析（Northern analysis），顯示EFE基因在乙烯誘導花瓣老化過程中扮 演重要角色。

Ethylene, a plant hormone, is known to play an important role in a variety of physiological process, including fruit ripening and flower senescence. Ethylene-forming enzyme(EFE), which converts 1-aminocyclopropane-l-carboxylic acid(ACC) to ethylene, is the key enzyme of ethylene biosynthesis in plants. Here we show the cDNA cloning and gene expression is relation to petal senescence for EFE of Phalaenopsis. Two conserved amino acid regions (NYPPCP and YPKFVF), based on a comparison of EFE amino acid sequences derived from various plants, was selected to generate two degenerate oligonucleotide primers. Using these primers and genomic DNA from Phalaenopsis (Lucky Lady X Pinlong Cardinal) as template, polymerase chain reaction (PCR)-based amplification was carried out. The PCR products were sequenced to confirm their homology to other EFE from various plants and used to screen a cDNA library, which was constructed using poly(A)⁺ RNA from senescing petals of Palaenopsis. One of the EFE cDNA clones (pPEFEA) was completely sequenced and characterized. The pPEFEA contained an open reading frame of 1349 base pairs encoding a sequence of 326 amino acids corresponding to 37.1 kDa polypeptide. The predicted amino acid sequence of pPEFEA was 70~72% identical to the deduced amino acid sequences of tomato EFE (pTOM13) and EFE-relaied genes isolated from avocado fruits and senescent carnation petals. The putative protein structure of pPEFEA posseses a N-glycosylation site between amino acid 113 to 142, and a conserved region of leucine zipper in an amphipathic α-helix region. Furthermore, the protein contains twelve conserved residues found in various enzymes which need ferrous ion and ascorbic acid as cofactors. Southern analysis indicated that pPEFEA belongs to single or low copy gene in Phalaenopsis genome. Northern analysis of ethylene-treated flowers indicated ethylene-forming enzyme plays an important role in ethylene-induced flower senescence.