堆肥未培养细菌的宏基因组文库构建及新的木聚糖酶基因的克隆和鉴定  

The Construction of A Metagenomic Library of Uncultured Bacteria from Compost and the Cloning and Identification of Novel Xylanase Genes

作　　者：张鹏[1] 段承杰[1] 庞浩[2] 封毅[2] 靳振江[2] 许跃强[2] 莫新春[2] 唐纪良[1] 冯家勋[1] 

机构地区：[1]广西亚热带生物资源保护利用重点实验室(广西大学) [2]广西大学生命科学与技术学院,广西南宁530005

出　　处：《广西科学》2005年第4期343-346,352,共5页Guangxi Sciences

基　　金：国家863计划项目(2004AA214140);国际科技合作重点项目计划(2002AA217121)资助项目

摘　　要：从自制堆肥样品中提取未培养细菌的总DNA,用柯斯质粒pW EB∷TNC为载体构建宏基因组文库,对文库进行筛选获得表达木聚糖酶活性的克隆,再进行亚克隆、测序分析以及B lastx搜索G enB ank分析木聚糖酶基因。结果构建得到一个包含约5万个克隆的宏基因组文库,文库中外源DNA总容量约为1.8×106kb,获得2个表达木聚糖酶活性的克隆:pGXN 1050和pGXN 1051,鉴定分析表明:pGXN 1050上潜在的木聚糖酶基因um xyn 11A具1个771bp的ORF(Open R ead ing F ram e),可编码含257个氨基酸的蛋白质,所编码产物与混合纤维弧菌(C ellv ibr io m ix tus)的内切-1,4-β-木聚糖酶(G enB ank索引号Z48925.1)的氨基酸序列有46%的一致性和57%的相似性;pGXN 1051上潜在的木聚糖酶基因um xyn 11B具一个723bp的ORF,可编码含241个氨基酸的蛋白质,编码产物与混合纤维弧菌的内切-1,4-β-木聚糖酶(G enB ank索引号Z48925.1)的氨基酸序列有73%的一致性和80%的相似性。木聚糖酶Um xyn11A和Um xyn11B都属于糖基水解酶家族11的成员。Metagenomic DNA of uncultured bacteria from self-made compost was extracted, endblunted and ligated with cosmid vector of pWEB∷TNC. A metagenomic library containing about 50,000 clones was constructed. The capacity of foreign DNA cloned in the library was 1. 8 × 10^6Kb. The library was screened for xylanase activity and two positive clones were isolated which were named as pGXN1050 and pGXN1051, respectively. The xylanase genes on the two clones were subcloned , sequenced and analysed. Results showed that the putative xylanase gene umxyn11A on pGXN1050 had a 771bp ORF, encoding a product with 257 amino acids sharing 46% identity and 57% similarity with endo-1,4-beta-xylanase（GenBank accession No. Z48925.1） of CeUvibrio mixtus. The putative xylanase gene umxyn11B on pGXN1051 had a 723bp ORF, encoding a product with 241 amino acids sharing beta-xylanase（GenBank accession No. Z48925.1） 73% identity and 80% similarity with endo-1,4- of Cellvibrio mixtus. Umxyn11A and Umxyn11B are members of family 11 glycosyl hydrolase.

关 键 词：细菌 宏基因组文库 木聚糖酶基因 

分 类 号：N242[自然科学总论]