酿酒酵母乙酸耐受基因的定位、筛选及初步验证
发布时间：2018-11-02 08:52
【摘要】：燃料乙醇作为一种绿色的生物质能源,具有很大的发展空间。在实际生产中通常使用酵母发酵纤维素水解液来生产乙醇。然而该水解液成分复杂,含有较多的抑制物───如乙酸、苯酚、糠醛等,这些物质均会影响发酵效能。乙酸作为主要的抑制物,可以引起酿酒酵母染色质沿核膜凝集、DNA断裂、氧化胁迫应激反应增加、线粒体膜去极化,从而使得酵母生长变慢甚至引起细胞的程序性死亡。虽然已有组学研究发现了部分候选基因,并且通过单基因过表达或者敲除技术增强了酿酒酵母的乙酸耐受性,但是关于酿酒酵母详细的乙酸耐受机制并未阐明,而且一些研究结果并不一致。因此,目前需要一种能够直接反映酵母本质变化的方法来对酿酒酵母的乙酸耐受性进行深入分析。本研究从酿酒酵母的基因层面分析,为研究酵母乙酸耐受性提供了一种可行的研究方法,并且该方法还适用于其他耐受机制的研究。本文研究对象为两株乙酸耐受表型差异显著的酿酒酵母单倍体───乙酸耐受菌株YHA及乙酸敏感菌株YLA。通过子代群体分析,实验室前期筛选获得三个与乙酸耐受相关的数量性状位点(quantitative trait loci,QTL)。精细查找三个QTL内的微卫星位点(simple sequence repeats,SSR)并筛选分析,获得41个SSR位点。通过毛细管电泳分析SSR在两个酿酒酵母单倍体YLA和YHA中的分布,发现仅有十个位点在两菌株间具有多态性。将这些SSR位点在38子代群体进行PCR扩增,进行毛细管电泳分析,该方法仅进一步缩小了12号染色体上的QTL区间,却未能进一步缩小9号、16号染色体上的QTL区间。结果表明SSR不能对QTL区间进行精细划分。全基因组重测序获得YHA及YLA的基因信息,对存在于三个QTL区间内的单核苷酸多态性(single nucleotide polymorphism,SNP)进行分类整理,通过对比两菌株之间的基因差异,发现在候选区间内两个菌株中分别存在78个和45个SNP。随后进行基因功能分类、蛋白质功能突变预测等生物信息学的分析,最终获得ECI1、FRE1、HOP1、IRC20、MAM33、PRK1、THI7、UTP25、YIR007W、YSH1等十个乙酸候选耐受基因。实验结果表明,通过SNP分析可以进一步缩小QTL基因区间,甚至可以鉴别出乙酸候选基因。对这十个基因在YHA菌株中进行单基因敲除的初步验证。实验结果发现,与单倍体菌株YHA相比,单基因缺失菌株?fre1乙酸耐受性下降10 mM,FRE1的基因编码一种铁离子还原酶和铜离子还原酶。而与此相反,单倍体菌株?prk1的乙酸耐受性增强10mM,PRK1基因编码一种蛋白质丝氨酸/苏氨酸激酶。实验表明结合SSR与SNP分子标记,可以利用较少的成本与信息鉴定出酿酒酵母乙酸耐受基因。并且本方法为研究酵母应对其他环境胁迫机制的研究提供了新思路。
[Abstract]:As a kind of green biomass energy, fuel ethanol has great development space. Yeast fermentation of cellulose hydrolysate is usually used to produce ethanol in actual production. However, the hydrolysate is complex in composition and contains more inhibitors such as acetic acid, phenol, furfural and so on, all of which affect fermentation efficiency. Acetic acid as the main inhibitor can cause yeast chromatin agglutination along the nuclear membrane, DNA breakage, oxidative stress response increase, mitochondrial membrane depolarization, which makes yeast growth slow and even cause programmed cell death. Although some candidate genes have been identified in previous studies and the acetic acid tolerance of Saccharomyces cerevisiae has been enhanced by single gene overexpression or knockout techniques, the detailed acetic acid tolerance mechanism of Saccharomyces cerevisiae has not been clarified. And some studies are inconsistent. Therefore, it is necessary to analyze the acetic acid tolerance of Saccharomyces cerevisiae directly. From the gene level of Saccharomyces cerevisiae, this study provides a feasible method for the study of acetic acid tolerance in yeast, and it is also suitable for the study of other tolerance mechanisms. In this paper, two acetic acid tolerant strains of Saccharomyces cerevisiae, YHA and YLA., which have different phenotypes of acetic acid tolerance, were studied. Three quantitative trait loci (quantitative trait loci,QTL) related to acetic acid tolerance were obtained by pre-laboratory screening. The microsatellite loci in three QTL (simple sequence repeats,SSR) were carefully searched and analyzed, and 41 SSR loci were obtained. The distribution of SSR in two haploid YLA and YHA of Saccharomyces cerevisiae was analyzed by capillary electrophoresis. Only 10 loci were found to be polymorphic between the two strains. These SSR loci were amplified by PCR in 38 progenies and analyzed by capillary electrophoresis. This method only further reduced the QTL interval on chromosome 12, but failed to further reduce the QTL interval on chromosome 9 and 16. The results show that SSR can not fine partition the QTL interval. The gene information of YHA and YLA was obtained by whole genome resequencing. Single nucleotide polymorphism (single nucleotide polymorphism,SNP (SNP), which existed in three QTL regions, was classified and sorted, and the genetic differences between the two strains were compared. 78 and 45 SNP. strains were found in the candidate interval, respectively. Then the bioinformatics analysis such as gene function classification protein function mutation prediction and so on were carried out. Finally ten acetic acid candidate tolerance genes such as ECI1,FRE1,HOP1,IRC20,MAM33,PRK1,THI7,UTP25,YIR007W,YSH1 were obtained. The results showed that SNP analysis could further narrow the interval of QTL gene and even identify the candidate gene of acetic acid. The single gene knockout of these ten genes in YHA strain was preliminarily verified. Compared with the haploid strain YHA, the single gene deletion strain, fre1, whose tolerance to acetic acid was reduced by 10 mM,FRE1, encoded a kind of iron ion reductase and copper ion reductase. In contrast, the acetic acid tolerance of haploid strain, prk1, was enhanced by the 10 mMmPPRK1 gene encoding a protein serine / threonine kinase. The results showed that the acetic acid tolerance gene of Saccharomyces cerevisiae could be identified by combining SSR and SNP molecular markers with less cost and information. This method provides a new idea for the study of yeast response to other environmental stress mechanisms.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933
本文编号：2305537
[Abstract]:As a kind of green biomass energy, fuel ethanol has great development space. Yeast fermentation of cellulose hydrolysate is usually used to produce ethanol in actual production. However, the hydrolysate is complex in composition and contains more inhibitors such as acetic acid, phenol, furfural and so on, all of which affect fermentation efficiency. Acetic acid as the main inhibitor can cause yeast chromatin agglutination along the nuclear membrane, DNA breakage, oxidative stress response increase, mitochondrial membrane depolarization, which makes yeast growth slow and even cause programmed cell death. Although some candidate genes have been identified in previous studies and the acetic acid tolerance of Saccharomyces cerevisiae has been enhanced by single gene overexpression or knockout techniques, the detailed acetic acid tolerance mechanism of Saccharomyces cerevisiae has not been clarified. And some studies are inconsistent. Therefore, it is necessary to analyze the acetic acid tolerance of Saccharomyces cerevisiae directly. From the gene level of Saccharomyces cerevisiae, this study provides a feasible method for the study of acetic acid tolerance in yeast, and it is also suitable for the study of other tolerance mechanisms. In this paper, two acetic acid tolerant strains of Saccharomyces cerevisiae, YHA and YLA., which have different phenotypes of acetic acid tolerance, were studied. Three quantitative trait loci (quantitative trait loci,QTL) related to acetic acid tolerance were obtained by pre-laboratory screening. The microsatellite loci in three QTL (simple sequence repeats,SSR) were carefully searched and analyzed, and 41 SSR loci were obtained. The distribution of SSR in two haploid YLA and YHA of Saccharomyces cerevisiae was analyzed by capillary electrophoresis. Only 10 loci were found to be polymorphic between the two strains. These SSR loci were amplified by PCR in 38 progenies and analyzed by capillary electrophoresis. This method only further reduced the QTL interval on chromosome 12, but failed to further reduce the QTL interval on chromosome 9 and 16. The results show that SSR can not fine partition the QTL interval. The gene information of YHA and YLA was obtained by whole genome resequencing. Single nucleotide polymorphism (single nucleotide polymorphism,SNP (SNP), which existed in three QTL regions, was classified and sorted, and the genetic differences between the two strains were compared. 78 and 45 SNP. strains were found in the candidate interval, respectively. Then the bioinformatics analysis such as gene function classification protein function mutation prediction and so on were carried out. Finally ten acetic acid candidate tolerance genes such as ECI1,FRE1,HOP1,IRC20,MAM33,PRK1,THI7,UTP25,YIR007W,YSH1 were obtained. The results showed that SNP analysis could further narrow the interval of QTL gene and even identify the candidate gene of acetic acid. The single gene knockout of these ten genes in YHA strain was preliminarily verified. Compared with the haploid strain YHA, the single gene deletion strain, fre1, whose tolerance to acetic acid was reduced by 10 mM,FRE1, encoded a kind of iron ion reductase and copper ion reductase. In contrast, the acetic acid tolerance of haploid strain, prk1, was enhanced by the 10 mMmPPRK1 gene encoding a protein serine / threonine kinase. The results showed that the acetic acid tolerance gene of Saccharomyces cerevisiae could be identified by combining SSR and SNP molecular markers with less cost and information. This method provides a new idea for the study of yeast response to other environmental stress mechanisms.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933
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相关博士学位论文 前2条
1 丁明珠;酵母对纤维素水解液中复合抑制剂耐受的系统分析与解耦[D];天津大学;2011年
2 胡小华;酵母数量性状主效基因的定位分离及克隆[D];复旦大学;2005年
相关硕士学位论文 前1条
1 肖银;酿酒酵母抗乙酸胁迫性状的数量性状基因座定位[D];江南大学;2015年
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