以聚合酶連鎖反應法選殖苦瓜ACC合成酶基因片段之研究

Cloning of Fragments of the Gene Encoding ACC Synthase from Bitter Gourd Utilizing Polymerase Chain Reaction

以苦瓜基因組DNA （genomic DNA）為模板，用蛋白質演化保留區為引子（primer），經聚合酶連鎖度應法（polymerase chain reaction）合成出四個苦瓜ACC合成酶（l-aminocyclopropane-l-carboxylate synthase）基因片段－－MAC11、MAC16、MAC17及MAC19。此四個基因片段經選殖入pUC18載體內，並經核苷酸定序分析，得知MAC11、MAC16、MAC17與MAC19分別具有259、253、253與242個鹼基對。將MACl1、MAC16、 MAC17及MAC19與其他作物ACC合成酶基因之核苷酸序列比較，其中分別含有一段109、103、100與91個鹼基對長度之隱子（intron），而可轉譯出之胺基酸分別為46、46、47與46個。在蛋白質相似性程度上，彼此間具有、43.5%～78.3%之同源程度，而與其他作物之ACC合成酶胺基酸序列比較，則具有43.5%～82.6%之同源程度。所以，在苦瓜中至少有四個ACC合成酶基因存在。所得到的西個苦瓜ACC合成酶基因片段，經由交叉雜交分析（cross hybridization），可知此四個片段可作為未來選殖cDNA及基因表現分析之專一性探針；且經由苦瓜基因組南方氏分析（genornic Southern analysis），得知此四個基因屬於單一拷貝（single copy）或低拷貝（low copy）基因，其中MAC11與MAC19可能位於染色體相同位置上。

Four gene fragments of 1- aminocyclopropane -1- carboxylate (ACC) synthase (EC 4. 1, 1. 14), the key regulatory enzyme catalyzing S -adenosylmethionine to ACC in the ethylene biosynthetic pathway, were cloned from bitter gourd (Momordica charantia L.). For this cloning the polymerase chain reaction was conducted with primers designed based on highly conserved regions of ACC synthase among different plant species. The sequences of these gene fragments (MAC11, MAC16, MAC17, and MAC19) have been determined and their respective length are 259, 253, 253, and 242 bp, of which introns are 109, 103, 100, and 91 bp. The open reading frames for these fragments contain 138, 138, 141, and 138 bp, respectively, encoding 46, 46, 47, and 46 amino acids. The degree of homology ranges from 43,5% to 78,3% at the protein level among these four clones, and from 43.5% to 82.6% by comparing the deduced amino acid sequences with other ACC synthase from different plant species. Cross hybridization one another showed that these four clones can serve as the specific probes for gene expression studies. Cfenomic DNA gel blot analysis indicated that the copy number for the corresponding genes is single or low in the genome of bitter gourd, and that MAC11 and MAC19 possibly link in the genome.