云南大额牛瘤胃宏基因组Fosmid文库的构建与分析

›› 2013, Vol. 40 ›› Issue (12): 61-65.

云南大额牛瘤胃宏基因组Fosmid文库的构建与分析

李碧凤¹, 朱雅新², 苟潇¹, 冷静¹, 毛华明¹, 李清¹, 冯励¹, 杨舒黎¹

1. 云南农业大学动物营养与饲料科学重点实验室, 云南昆明 650201;
2. 中国科学院微生物研究所, 北京 100101

收稿日期:2013-07-04 出版日期:2013-12-20 发布日期:2014-02-11
通讯作者: 杨舒黎 E-mail:yangshuli77@163.com
作者简介:李碧凤（1986- ），女，云南人，硕士生，研究方向：反刍动物肠道微生态与兽医寄生虫学。
基金资助:
国家自然科学基金（31160467、31360562、30960256、31060314、31160449、31260543）；云南省自然基金（2010CD059、2005C00038）；“863”计划重点项目（2008AA101001）。

Construction and Analysis of Metagenomic Fosmid Library from Rumen in Yunnan Gayal

LI Bi-feng¹, ZHU Ya-xin², GOU Xiao¹, LENG Jing¹, MAO Hua-ming¹, LI Qing¹, FENG Li¹, YANG Shu-li¹

1. Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming 650201, China;
2. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

Received:2013-07-04 Online:2013-12-20 Published:2014-02-11

摘要/Abstract

摘要： 为研究云南大额牛瘤胃中丰富的降解纤维素类物质的基因，采用包埋法和脉冲场电泳技术直接从云南大额牛瘤胃内容物提取总DNA，经不完全酶切获得DNA片段，大小为23~48 kb，与pcc2 FOS vector连接，转化至E.coli EPI 300宿主细胞，构建瘤胃微生物宏基因组Fosmid文库。该文库共保存38400个克隆，平均插入片段大小约35.5 kb，空载率小于2%，库容达1363 Mb。通过对该文库进行部分纤维素酶活力筛选，获得具有羧甲基纤维素酶活性的克隆95个，木聚糖酶活性的克隆52个，同时具有这2种酶活性的克隆23个，这为进一步研究大额牛瘤胃内纤维素酶基因奠定了基础。

关键词: 大额牛; 瘤胃微生物; Fosmid文库; 筛选

Abstract: To study cellulose genes of Yunnan Gayal rumen,the high molecular weight DNA was extracted and purified from rumen contents of 4 adult Gayals by embedding method and pulsed field gel electrophoresis approaches. After digestion with AluⅠ, DNA fragments ranging from 23~48 kb were collected and ligated with pcc2 FOS vector and transformed into E.coli EPI 300.A rumen metagenomic Fosmid library with the capacity of 1363 Mb was successfully constructed. This library contained 38400 clones with an average inserting size of 35.5 kb,and empty rate was lower than 2%.The screening of the partial metagenomic library identified 95 clones expressing activity of carboxymethyl cellulase,52 clones with xylanase activity, and 23 clones with carboxymethyl cellulase and xylanase activity. These results provided the foundation to further study cellulase gene in Gayal rumen.

Key words: Gayal; rumen microbes; Fosmid library; screen

中图分类号:

S823

李碧凤, 朱雅新, 苟潇, 冷静, 毛华明, 李清, 冯励, 杨舒黎. 云南大额牛瘤胃宏基因组Fosmid文库的构建与分析[J]. , 2013, 40(12): 61-65.

LI Bi-feng, ZHU Ya-xin, GOU Xiao, LENG Jing, MAO Hua-ming, LI Qing, FENG Li, YANG Shu-li. Construction and Analysis of Metagenomic Fosmid Library from Rumen in Yunnan Gayal[J]. China Animal Husbandry & Veterinary Medicine, 2013, 40(12): 61-65.

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