为了解曲霉(Aspergillus sp.)N1-14’的高产L-苹果酸（LMA）机制，提取其总DNA作模板，设计同源引物扩增包含丙酮酸羧化酶基因（pyc）和苹果酸脱氢酶基因（mdh）全长的片段并测序，再参考测序结果找到丙酮酸羧化酶(PYC)和苹果酸脱氢酶(MDH)的编码区，设计引物从N1-14’总cDNA扩增出其编码序列并通过TA克隆测序。测序结果显示pyc基因编码区长3582 bp，编码1193aa，分析结果显示PYC氨基酸序列在曲霉属内相当保守，同源性高达90%以上，其中N1-14’在两个保守位点出现突变，833位的A位于一个环区和1022位的F位于α-螺旋中部，可能与其高产酸活性相关；mdh编码区全长1023 bp，编码340aa。MDH氨基酸序列高度保守，突变株同样有两个保守区出现氨基酸点突变，且两点均出现在α-螺旋区域。本试验主要克隆两个曲霉N1-14’产L-苹果酸通路关键酶基因，分析其种属的特异性及预测特异氨基酸位点的功能，为继续探究N1-14’的高产LMA机制及相应的基因工程改造提升产酸水平奠定基础。

In order to understand the mechanism behind the high production of L-malate (LMA) by Aspergillus sp. N1-14’, its total DNA was extracted to be used as the template, homologous primers were designed to amplify the segment containing the full length of the pyruvate carboxylase gene (pyc) and the malate dehydrogenase gene (mdh), and sequencing was then performed. Based on the results of this sequencing, the coding regions of pyc and mdh were identified, their coding sequences were amplified from the total cDNA of N1-14’ using the designed primers, and sequencing was carried out through TA cloning. The results of the sequencing showed that the open reading frame (ORF) of pyc had 3582 bp and it encoded 1193 amino acids. The analysis of the results showed that the amino acid sequence of pyruvate carboxylase (PYC) was quite conserved in Aspergillus species as the sequence similarity with other Aspergillus species was higher than 90 %. Mutations occurred at two conserved sites of N1-14’, 833(A) and 1022(F), which were located in a loop and in the middle of α-helix, respectively, suggesting that those mutations may be relevant to the high production of LMA. The ORF of mdh had 1023 bp and it encoded 340 amino acids. The amino acid sequence of malate dehydrogenase (MDH) was also conserved across the Aspergillus species, and there were two amino acid mutation sites in the conserved domain. Both of them were situated in the α-helix. In this experiment, two genes related to the key process of producing LMA in N1-14’ were cloned, the specificity of these two genes was analyzed, and the function of the specific amino acid sites were predicted. This study provides a basis for further study on the mechanisms behind the high production of LMA and genetic manipulations required for increasing its production.

国家自然科学基金项目（31271940）