蝴蝶蘭花瓣老化相關基因之選殖及分析

Cloning and Characterization of Phalaenopsis Genes Related to Petal Senescence

延長花卉花瓣的觀賞壽命一直是園藝界的重要課題，而授粉可迅速造成蝴蝶蘭花瓣的萎凋。為了瞭解花瓣老化的機制，本研究構築了一個蝴蝶蘭授粉後天之花瓣互補DNA庫（cDNA library），以專一性基因片段為探針，選殖得到下列三個基因，分別說明如次：（一）pOACS10：互補DNA全長為1,674個鹼基對（base pairs），具有一個長度為1,338個鹼基對之蛋白質開放讀碼框架（open reading frame），可解讀445個胺基酸，推演之分子量為49,666Da，等電點為6.55，經電腦搜尋序列資料庫的結果顯示，OACS10解碼的胺基酸序列系ACC合成酶。（二）pOACS77：全長共為1,590個鹼基對，可解讀445個胺基酸，分子量為50,033Da，等電點為5.85，亦為ACC合成酶，分析以上二演繹蛋白之全長胺基酸序列，顯示均含有pyridoxal-5'-phosphate及基質S-adenosyl-L-methionine鍵結位置，分別為Lys275及Lys272。（三）pOACO31：長為2,418個鹼基對的互補DNA，具有一個長度為2,100個鹼基對之蛋白質開放讀碼框架，可解讀699個胺其酸，分子量為78,032Da，等電點為8.74。經由電腦比對分析，發現此多胜肽並非ACC合成酶，而和人類、老鼠及酵母菌過氧化體中的乙醯輔酶A氧化酶（acyl-CoA oxidase）具較高的同源性，並具有共同之活性部位－－黃素單核苷酸（flavin mononucleotide）結合區。在基因表現方面，外加乙烯處理蝴蝶蘭花瓣不同時間，無法偵測至對應於pOACS10之mRNA的累積，pOACO31基因於0.5小時內遞減，並於1小時後，隨著時間的增加增強，pOACS77基因則於3小時開始表現，在24小時達到高峰。以專一性的基因片段探針，進行基因組南方氏雜交分析，結果顯示上述三個基因分別位於基因組的不同位置，且為單一或低拷貝。

The control of flower longevity is of great interest for horticulturists. In Phalaenopsis, pollination results in accelerated wilting of the corolla. As a first step towards understanding the mechanism of flower senescence, we isolated three cDNAs, named pOACS10, pOACS77 and pOACO31, from an cDNA library constructed by poly (A)(superscript +) RNA from Phalaenopsis petals at one day after pollination, pOACS10, 1,674 bp long, encodes a polypeptide of 445 amino acids with a predicted isoelectric point of 6.55 whose molecular weight is 49,666 Da pOACS77 is 1,590 bp encoding a sequence of 445 amino acids corresponding to a 50,033 Da polypeptide which has a predicted isoelectric point of 5.85. Based on computer analysis and sequence comparison, both polypeptides show highy homoiogy to ACC synthase. Both of them contain the active site on Lys275 and Lys272, respectively, that is proposed to bind the pyridoxal-5'-phosphate and covalently link to the S-adenosyl-L-methionine. The third cDNA pOACO31, whose molecular weight is 78,032 Da, encodes a polypeptide of 699 amino acids with an isoelectric point of 8.74. According to the searching of database, this polypeptide shows homology to peroxisomal acyl-CoA oxidase from human (Homo sapiens), rat (Rattus norvegicus), yeast (Candida tropicalis) and baker's yeast (Saccharomyces cerevisiae). The presence of a putative flavin mononucleotide-binding site and the conserved amino acid residues for acyl-CoA oxidases are found in an arrayed sequence manner in OACO31 polypeptide. These data strongly suggest that the pOACO31 encodes peroxisomal acyl-COA oxioase. Northern analysis showed that, after treatment of ethylene, the mRNA for pOACS77 started to accumulated at 3 hours and reached to a maximum at 24 hours. Upon treatment with ethylene, the mRNA accumulation for pOACO31 was decreased within 30 minutes and increased after 1 hour. Genomic Southern analysis indicates indicates that all there genes are located on different regions of chromosomes and belong to single or low copy gene in Phalaenopsis genome.