中文摘要：

根据转录组测序结果设计特异性引物,以深黄被孢霉（Mortierella isabellina）M6-22的c DNA为模板,PCR扩增苹果酸脱氢酶基因MIMDH2,测序结果显示该序列长1 017 bp,编码338个氨基酸。序列分析表明该序列与已报道的烟曲霉（Aspergillus fumigates）线粒体苹果酸脱氢酶的相似性最高,达71.26%,且含有苹果酸脱氢酶的保守辅酶结合位点、底物结合位点和催化活性位点。将MIMDH2片段连接到表达载体p ET32a（＋）中,构建重组表达质粒p ET32a-MIMDH2,并转化至大肠杆菌BL21中进行诱导表达,SDS-PAGE电泳检测结果显示在50 k D左右处有一蛋白质条带,酶活分析结果显示经镍柱亲和层析纯化的重组蛋白酶活高达271.33 U/mg。以上结果说明所克隆的MIMDH2为一个新的潜在的苹果酸脱氢酶基因,所编码的蛋白质具有苹果酸脱氢酶的活性。

英文摘要：

A pair of gene-specific primers was designed based on the RNA-seq data, and a full-length cD- NA fragment of 1 017 bp, designated MIMDH2, was obtained from Mortierella isabellina strain M6-22 by PCR amplification using cDNA as template. Sequence analysis showed that it comprised an open reading frame encoding 338 amino acids. The deduced amino acid sequence shared the highest identity of 71.26% with that of Aspergillus fumigates, and it also contained the conserved coenzyme and substrate binding sites, as well as catalytic active sites. The cDNA sequence was further subeloned into expression vector pET32a （＋） to generate recombinant plasmid pET32a-MIMDH2, which was subsequently transformed into Escherichia coli BL21 for inducing expression. The expressed protein was purified by Ni-NTA affinity chromatography. Enzymatic activity analysis demonstrated that the specific activity of the recombinant protein was 271.33 U/rag. All these results demonstrated that the amplified eDNA fragment MIMDH2 is a novel and potential malate dehydrogenase gene and its expressed protein has the catalytic activity of malate dehydrogenase.