高效产氢菌B49菌株adh和L-ldh基因克隆及序列分析

设计细菌adh基因和L-ldh基因简并引物, 采用降落PCR并结合Cassette PCR方法从高效产氢菌B49中扩增全长基因。共获得两段基因组DNA片段。其中一段序列长1902 bp, 包括adh基因开放阅读框1101 bp, 共编码366个氨基酸, 编码产物分子量为39.71 kD, 理论等电点为pH 5.93。该基因与Clostridium thermocellum ATCC 27405的adh位点序列一致性为73%。另一段序列长2490 bp, 包括L-ldh基因开放阅读框951 bp, 共编码316个氨基酸, 编码产物分子量为34.23 kD, 理论等电点为pH 6.09。该基因与Bacillus megaterium的L-ldh位点一致性为74%。试验结果表明, 采用CodeHop和Genefisher程序化设计的简并引物可信性强, 阳性率高, CodeHop设计简并引物效果要好于Genefisher。adh和L-ldh的成功克隆为通过代谢工程手段敲除adh和L-ldh基因提供了科学依据, 从而为进一步提高B49产氢能力的代谢工程研究奠定基础。

Clone the Alcohol Dehydrogenase and L-lactate Dehydrogenase of Bio-hydrogen Producing Bacteria Ethanoligenens harbinense B49 and Its Sequence Analysis

Designed the degenerate primers of alcohol dehydeogenase and L-lactate dehydrogenase to augment Ethanoligenens harbinense B49 genomic DNA, and obtained about 780 bp and 610 bp PCR product respectively. Augmented flank sequences of the two PCR fragments with the Cassette PCR method. Similarity alignment showed that the products of the cloned DNA were very high similar to those of alcohol dehydrogenase genes and L-lactate dehydrogenase genes respectively. One of the two sequences was 1902 bp long, and the ORF of adh was 1101 bp long and encoded 366 amino acids. Its putative molecular weight was about 39.71 kD, its calculational isoionic point was pH 5.93. The maximal identity and positive was 51% and 73% with Clostridium thermocellum ATCC 27405 adh. The other one was 2490 bp long, and the ORF of adh was 951 bp long and encoded 316 amino acids. Its putative molecular weight was 34.23 kD, its calculational isoionic point was pH 6.09. The maximal identity and positive was 55% and 74% with Bacillus megaterium L-ldh. Successfully cloning these two genes would not only enrich the gene resources of L-lactate dehydrogenase and alcohol dehydrogenase genes, but also give the scientific warrant for the metabolic engineering research and the construction of the gene-engineering bacteria.