深海沉积物宏基因组文库构建及淀粉酶筛选  

Construction of metagenomic DNA library of deep-sea sediments and screening of amylase

作　　者：黄雅丽[1,2] 张炯 曹理想[2] 谭红铭[2] 陆勇军[2] 周世宁[2] 

机构地区：[1]广州中医药大学基础医学院,广东广州510006 [2]中山大学生命科学院生物防治国家重点实验室,广东广州510275 [3]深圳华大基因研究院,广东深圳518083

出　　处：《海洋学报》2013年第1期144-148,共5页

基　　金：国家高科技研究与开发(863)项目(2008AA09Z402);大洋探索项目(DYXM-115-02;2;14)

摘　　要：深海环境通常具有高盐,高压,高/低温,无光照等特点,使得海洋微生物存在一套独特的生理代谢机制和分子细胞结构,然而迄今绝大部分深海微生物不能在实验室条件下被分离培养,深海微生物资源开发遇到很大挑战。本研究通过不依赖培养的方法研究海洋微生物的基因资源,构建了南海深海沉积物fosmid宏基因组文库,共获得约39 600个克隆,插入片段范围在24～45kb之间,平均插入片段大小为33kb,克隆片段的总库容达到1 320Mb。通过功能筛选获得3个具有淀粉酶活性的克隆子,选取其中最适温度较低的amy7作为进一步研究对象。构建amy7插入片段的重组质粒文库,获得一个同样有淀粉酶活性的克隆子amy7—6。经测序,克隆子amy7—6含有3 291bp插入片段,序列比对分析后发现其中一个大小为1 920bp的ORF,其编码的蛋白质序列(AmyS)与各种来源的糖苷酶有着较高的相似性。Deep sea environment is usually characterized by extremely high salinity, high pressure, high/low tem- perature and darkness. The physicochemical features ensured that may exist in microorganisms unique metabolic and physiological activities, molecular and cellular structure to adapt to these extreme conditions. Therefore, the exploration of biological and function diversity of microorganisms in deep sea are expected to yield novel metabo- lites. To access to the microbial genetic resources of deep-sea sediment with a culture-independent approach, The sediment DNA was extracted from deep sea sediments of the South China Sea and cloned into fosmid vector genera- ting a library of 39 600 clones with inserts of 24-45 kb and an average insert size of 33 kb. The metagenomic li- brary represented about 1 320 Mbp of genomic DNA in deep-sea sediments. The library was screened for amylase activity and three recombinant clones showed amylolytic activity. The clone, amy7, with lower optimal tempera- ture was further analyzed. Subsequent library of insert DNA of amy7 was constructed and a positive subclone, des- ignated as amyT-4, was screened. Sequencing of the clone revealed that it contains a 3 291 bp fragment. An open reading frame of 1 920 bp was identified from the fragment and showed high identity with genes of glycosidase from various organisms.

关 键 词：深海沉积物 fosmid宏基因组文库 海洋微生物 淀粉酶 

分 类 号：Q934[生物学—微生物学]