Construction of metagenomic DNA library of deep-sea sediments and screening of amylase
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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24~45 kb之间,平均插入片段大小为33 kb,克隆片段的总库容达到1 320 Mb。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3 291 bp插入片段,序列比对分析后发现其中一个大小为1 920 bp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。
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关键词:
- 深海沉积物 /
- fosmid宏基因组文库 /
- 海洋微生物 /
- 淀粉酶
Abstract: Deep sea environment is usually characterized by extremely high salinity, high pressure, high/low temperature 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 metabolites. 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 generating a library of 39 600 clones with inserts of 24-45 kb and an average insert size of 33 kb. The metagenomic library 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 temperature was further analyzed. Subsequent library of insert DNA of amy7 was constructed and a positive subclone, designated as amy7-4, 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.-
Key words:
- deep-sea sediment /
- fosmid metagenomic library /
- marine microorganisms /
- amylase
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