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张鹏,段承杰,庞浩,封毅,靳振江,许跃强,莫新春,唐纪良,冯家勋.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[J].广西科学,2005,12(4):343-346,352. [点击复制]
- Zhang Peng,Duan Chengjie,Pang Hao,Feng Yi,Jin Zhenjiang,Xu Yueqiang,Mo Xinchun,Tang Jiliang,Feng Jiaxun.The Construction of A Metagenomic Library of Uncultured Bacteria from Compost and the Cloning and Identification of Novel Xylanase Genes[J].Guangxi Sciences,2005,12(4):343-346,352. [点击复制]
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| 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 |
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张鹏1,2,3, 段承杰1,2,3, 庞浩3, 封毅3, 靳振江3, 许跃强3, 莫新春3, 唐纪良1,2,3, 冯家勋1,2,3
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| (1.广西亚热带生物资源保护利用重点实验室(广西大学), 广西南宁 530005;2.微生物及植物遗传工程教育部重点实验室(广西大学), 广西南宁 530005;3.广西大学生命科学与技术学院, 广西南宁 530005) |
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| 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pWEB∷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Blastx搜索GenBank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umxyn 11A具1个771bp的ORF(Open Reading Frame),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(Cellvibrio mixtus)的内切-1,4-β-木聚糖酶(GenBank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umxyn 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(GenBank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Umxyn11A和Umxyn11B都属于糖基水解酶家族11的成员。 |
| 关键词: 细菌 宏基因组文库 木聚糖酶基因 |
| DOI： |
| 投稿时间：2005-04-18修订日期：2005-05-19 |
| 基金项目:国家863计划(2004AA214140)和国际科技合作重点项目计划(2002AA217121)资助项目。 |
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| The Construction of A Metagenomic Library of Uncultured Bacteria from Compost and the Cloning and Identification of Novel Xylanase Genes |
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Zhang Peng1,2,3, Duan Chengjie1,2,3, Pang Hao3, Feng Yi3, Jin Zhenjiang3, Xu Yueqiang3, Mo Xinchun3, Tang Jiliang1,2,3, Feng Jiaxun1,2,3
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| (1.Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization at Guangxi Univ., Nanning, Guangxi, 530005, China;2.The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engi. at Guangxi Univ., Nanning, Guangxi, 530005, China;3.Coll. of Life Sci. and Tech., Guangxi Univ., Nanning, Guangxi, 530005, China) |
| Abstract: |
| Metagenomic DNA of uncultured bacteria from self-made compost was extracted,end-blunted 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 1.8×106Kb.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 pGXN 1050 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 Cellvibrio mixtus. The putative xylanase gene umxyn 11B on pGXN1051 had a 723bp ORF, encoding a product with 241 amino acids sharing 73% identity and 80% similarity with endo-1, 4-beta-xylanase(GenBank accession No.Z48925.1) of Cellvibrio mixtus. Umxyn 11A and Umxyn 11B are members of family 11 glycosyl hydrolase. |
| Key words: bacteria metagenomic library xylanase gene |
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