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深海沉积物宏基因组文库构建及淀粉酶筛选

DOI: 10.3969/j.issn.0253-4193.2013.01.016, PP. 144-148

Keywords: 深海沉积物,fosmid宏基因组文库,海洋微生物,淀粉酶

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Abstract:

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39600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1320Mb。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3291bp插入片段,序列比对分析后发现其中一个大小为1920bp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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