莆田豆梨 GPX 基因克隆与生物信息学分析

摘要

采用同源克隆 RT-PCR 结合 RACE 技术，从福建省地方梨种质资源莆田豆梨中克隆出 GPX 基因的cDNA 全长序列分离克隆并运用生物信息学方法对序列进行分析。克隆得到福建省地方种质资源莆田豆梨 GPX基因932 bp 的 cDNA 全长序列，分析表明，该序列的5′端和3′端的非编码序列长度分别为204 bp 和221 bp，开放阅读框为507 bp，编码168个氨基酸。氨基酸序列与苹果、柑橘、龙眼、荔枝的同源性90％以上。GPX 基因在 GenBank 上登录，登录号为 JQ011278。生物信息学分析表明：GPX 蛋白的分子量是20083．0 Da，理论等电点 pI 5．61，是不具跨膜结构域的亲水性胞质蛋白，不具有信号肽，细胞主要定位在细胞质上，有1个区域最有可能形成卷曲螺旋，由26．58％的 a 螺旋，19．26％的延伸链和54．16％的不规则卷曲组成，磷酸化位点有12个。此外，还对 GPX 酶分子三维立体结构等进行预测和分析。%Aglutathione peroxidasegene (GPX)was cloned from the leaves of Putian pear,PyruscalleryanaDecne. The RT-PCR and RACE were applied to obtain the complete cDNA sequence for GPX .The full length of the cDNA was approximately 932 bp consisting of an open reading frame of 507 bpwith the 5′-and 3′-untranslated regions of 204 bp and 221 bp,respectively.The putative protein had 168 amino acids and was greater than 90% homogenous on the sequence with Maluspumila,Citrus reticulata Banco,Dimocarpuslongan and Litchi chinensis .The gene had been registered in GenBankwith a code of JQ011278.The nucleotide and amino acid sequences of GPX were analyzed by using a bioinformatics software.The results showed that the protein had a molecular weight of 20 083.0 Da anda theoretical pI 5.61 .It was a hydrophilic cytoplasmic protein without transmembrane domain and signal peptide.The cell was mainly located in the cytoplasm at a region most likely to form coiled-coilcontaining 26.58% α-spiral,19.26% extending chain and 54.16% irregular curl.And,there were 12 phosphorylation sites on the cell.A3-Dmolecular structure of the enzyme was proposed with relevant analysis.