碱性果胶裂解酶基因的克隆及核苷酸序列分析

碱性果胶裂解酶基因的克隆及核苷酸序列分析 Cloning and Sequencing of a Alkaline Pectate Lyase Gene 翟超 ZHAI Chao 1 first-author 曹军卫 CAO Jun\|wei 1 武汉大学生命科学学院,湖北,武汉,430072 武汉大学生命科学学院,湖北,武汉,430072 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 以 p Bluescript KS（M13- ）质粒作为载体构建嗜碱芽孢杆菌 NTT33的基因组文库 .在 YC平板上筛选到一株含果胶裂解酶 （pectate lyase,pel）基因的大肠杆菌阳性克隆子 .酶切后 ,电泳鉴定 ,插入的外源片段约 6 .5kb.通过引物步行法测定了含有该基因的约 3kb左右的外源 DNA片段的核苷酸序列 .经 PCgene软件分析发现第 15 0～ 116 0 bp编码一个由 337个氨基酸组成的蛋白质分子 .根据果胶裂解酶的等电点分类方法推断 ,其属于Pel A.将其与 Genebank中现有的基因编码的氨基酸序列进行同源比较 ,发现它与 Bacillus sp.Ksm SM- P15的Pel E和 Azospirillum irakense的 Pel A的同源性分别为 38%和 32 % .这 3种酶与其它果胶裂解酶的基因同源性很低 ,所以推测它们很可能属于一个新的果胶裂解酶家族 A pectate lyase gene was cloned from genome library of Bacillus alcalophillus NTT33. A positive clone was screened in YC plate. A about 6.5 kb DNA fragment containing pectate lyase gene was sequenced. According to the analysis in PCgene,it was found that DNA sequence from 150\|1 160 bp was a ORF region coding 337 amino acid of a pectate lyase. The recombinant plasmid was identified by restriction enzyme analysis. This PelA had some regions that were homologous to a PelE from an alkaliphilic strain of Bacillus with 38% identity and a PelA from Azospirilluum irakense with 32% identity.So it was supposed to be a new pectate lyase gene.The predict pI of the Pel was 5.3,so it belonged to pelA superfamily according to the established Pel superfamily. 碱性果胶裂解酶 嗜碱芽孢杆菌 克隆 核苷酸序列分析 alkaline petate lyase Bacillus alcalophillus gene cloning nucleotide sequcucing Q785 2001-02-01 2021-04-01 2