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首页> 外文期刊>African Journal of Biotechnology >Rapid approach for cloning bacterial single-genes directly from soils
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Rapid approach for cloning bacterial single-genes directly from soils

机译：直接从土壤中克隆细菌单基因的快速方法

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Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of cloning strategy in this study. Five?GS?genes were directly cloned from soils using degenerate primers with two steps of nested polymerase chains reactions. The genes showed 94 to 99% amino acid identities to the homologs in the known database, and encoded proteins affiliated to GS I and GS II families, respectively. All the five genes could rescue the growth of?Escherichia coli?glutamine auxotroph mutant ET6017 in minimum medium (ammonium chloride was sole nitrogen source in this medium). This study develops one rapid approach for cloning bacterial single-genes directly from soils. Comparing with the conventional strategies for gene cloning from complex environmental samples, this method did not need making genomic library and isolating target genes from large amount of library clones. This approach distinctively demonstrates its advantages of rapidity and effectiveness particularly when it aims at cloning short single-genes that had known homologs in all kinds of nucleic acid databases.
机译：从环境样品中获取细菌的功能基因通常取决于基于库的方法，这种方法因其工作量大，阳性克隆可能性小而不受青睐。以一种细菌单基因编码谷氨酰胺合成酶（GS）为例，以检测克隆策略的效率。使用简并引物通过两个步骤的嵌套式聚合酶链反应，从土壤中直接克隆了5个GS基因。这些基因与已知数据库中的同系物显示94至99％的氨基酸同一性，并分别编码与GS I和GS II家族相关的蛋白质。这五个基因都可以在最小培养基（氯化铵是该培养基唯一的氮源）中拯救大肠杆菌-谷氨酰胺营养缺陷型突变体ET6017的生长。这项研究开发了一种直接从土壤中直接克隆细菌单基因的快速方法。与从复杂的环境样品中克隆基因的常规策略相比，该方法不需要建立基因组文库和从大量文库克隆中分离目标基因。该方法特别证明了其快速性和有效性的优势，特别是当其目的是克隆在各种核酸数据库中具有已知同源物的短单基因时。

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