曲霉N1-14’高产L-苹果酸相关基因的克隆与分析

发布时间：2018-05-14 01:23

  本文选题：曲霉N-’ + PYC　； 参考：《现代食品科技》2017年02期

【摘要】：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。
[Abstract]:In order to understand the mechanism of high yield LMAs of Aspergillus sp. N1-14', the total DNA of Aspergillus sp. N1-14 'was extracted as template, and the fragments containing pyruvate carboxylase gene (PYC) and malate dehydrogenase gene (mdh) were amplified by homologous primers and sequenced. The coding regions of pyruvate carboxylase (Pyc) and malate dehydrogenase (MDH) were found by referring to the sequencing results. Primers were designed to amplify the coding sequence from N1-14'total cDNA and sequenced by TA cloning. The results of sequencing showed that the coding region of pyc gene was 3582 BP, encoding 1193A. The results showed that the amino acid sequence of PYC was conserved in Aspergillus, and the homology was over 90%. Among them, A of N1-14 'mutation at two conserved sites is located in a ring region and F at position 1022 is located in the middle of 伪 -helix, which may be related to its high acid activity. The coding region of mdh is 1023 BP in length. The amino acid sequence encoding 340aa.MDH is highly conserved, and the amino acid sequence of N1-14' is highly conserved. There were two amino acid point mutations in two conserved regions of the mutant, and both appeared in the 伪 -helix region. In this study, two key enzyme genes of L- malic acid pathway produced by Aspergillus N1-14 'were cloned, and their species-specificity and function of predicting specific amino acid sites were analyzed. The results laid a foundation for further study on the mechanism of high yield LMA of N1-14 'and the corresponding genetic engineering to improve acid production level.
【作者单位】： 中国科学院南海海洋研究所;广东省微生物研究所省部共建华南应用微生物国家重点实验室广东省菌种保藏与应用重点实验室广东省微生物新技术公共实验室;中国科学院大学;广东环凯微生物科技有限公司;
【基金】：国家自然科学基金项目(31271940)
【分类号】：TS202.3
，

本文编号：1885700