河套蜜瓜ACC氧化酶基因cDNA部分片段的克隆和序列分析

Molecular Cloning and Sequence Analysis of a cDNA Fragment Encoding ACC Oxidase Gene from Hetao Melon (Cucumis melo L.)

以河套蜜瓜(CucumismeloL.cvHetao)成熟果实为材料,用硫氰酸胍法提取总RNA,以oligo(dT)15为引物,反转录合成cDNA,利用一对甜瓜ACC氧化酶(1-aminocyclopropane-1-carboxylateoxidase,ACO)基因cDNA的特异性引物进行聚合酶链式反应(PCR)扩增,获得了预期大小的cDNA片段.将此片段克隆于pUC19质粒中,进行DNA序列分析.序列分析结果表明该片段为545bp,编码甜瓜ACO的第126至第306个氨基酸.该河套蜜瓜ACO基因cDNA序列与Andes甜瓜、Cantaloupcharentais甜瓜和哈密瓜的相应序列进行比较,其核苷酸序列与Andes甜瓜的同源性为99.4%;与Cantaloupcharentais甜瓜和哈密瓜的同源性均为99.6%,与其对应的氨基酸序列的同源性均为99.4%.

Total RNA was isolated from ripe fruits of Hetao melon (Cucumis melo L.)by acid guanidinium thiocyanate-phenol-chloroform extraction method.The first strand of cDNA was synthesized by a reverse transcription using oligo(dT)(15) as a primer.A cDNA fragment was obtained by PCR amplification with a pair of primers which are specific to melon 1-aminocyclopropane-1-carboxylate(ACC) oxidase gene cDNA.The cDNA fragment was cloned into Sma I site of vector pUC19 and the nucleotide sequence was analyzed.The results show that the cDNA consists of 545bp,which encodes the amino acid from 126 to 306 of melon ACC oxidase.The sequence comparison between this cDNA and the corresponding cDNA of Andes melon,Cantaloup charentais melon and Hami melon show respectively that the homology of nucleotide sequence between Hetao melon and Andes melon is 99.4%,and that between Hetao melon and Cantaloup charentais melon,Hami melon both are 99.6%.The homology of the whole amino acid sequences is 99.4%.