【摘要】：燃料乙醇作為一種綠色的生物質能源,具有很大的發展空間。在實際生產中通常使用酵母發酵纖維素水解液來生產乙醇。然而該水解液成分復雜,含有較多的抑制物───如乙酸、苯酚、糠醛等,這些物質均會影響發酵效能。乙酸作為主要的抑制物,可以引起釀酒酵母染色質沿核膜凝集、DNA斷裂、氧化脅迫應激反應增加、線粒體膜去極化,從而使得酵母生長變慢甚至引起細胞的程序性死亡。雖然已有組學研究發現了部分候選基因,并且通過單基因過表達或者敲除技術增強了釀酒酵母的乙酸耐受性,但是關于釀酒酵母詳細的乙酸耐受機制并未闡明,而且一些研究結果并不一致。因此,目前需要一種能夠直接反映酵母本質變化的方法來對釀酒酵母的乙酸耐受性進行深入分析。本研究從釀酒酵母的基因層面分析,為研究酵母乙酸耐受性提供了一種可行的研究方法,并且該方法還適用于其他耐受機制的研究。本文研究對象為兩株乙酸耐受表型差異顯著的釀酒酵母單倍體───乙酸耐受菌株YHA及乙酸敏感菌株YLA。通過子代群體分析,實驗室前期篩選獲得三個與乙酸耐受相關的數量性狀位點(quantitative trait loci,QTL)。精細查找三個QTL內的微衛星位點(simple sequence repeats,SSR)并篩選分析,獲得41個SSR位點。通過毛細管電泳分析SSR在兩個釀酒酵母單倍體YLA和YHA中的分布,發現僅有十個位點在兩菌株間具有多態性。將這些SSR位點在38子代群體進行PCR擴增,進行毛細管電泳分析,該方法僅進一步縮小了12號染色體上的QTL區間,卻未能進一步縮小9號、16號染色體上的QTL區間。結果表明SSR不能對QTL區間進行精細劃分。全基因組重測序獲得YHA及YLA的基因信息,對存在于三個QTL區間內的單核苷酸多態性(single nucleotide polymorphism,SNP)進行分類整理,通過對比兩菌株之間的基因差異,發現在候選區間內兩個菌株中分別存在78個和45個SNP。隨后進行基因功能分類、蛋白質功能突變預測等生物信息學的分析,最終獲得ECI1、FRE1、HOP1、IRC20、MAM33、PRK1、THI7、UTP25、YIR007W、YSH1等十個乙酸候選耐受基因。實驗結果表明,通過SNP分析可以進一步縮小QTL基因區間,甚至可以鑒別出乙酸候選基因。對這十個基因在YHA菌株中進行單基因敲除的初步驗證。實驗結果發現,與單倍體菌株YHA相比,單基因缺失菌株?fre1乙酸耐受性下降10 mM,FRE1的基因編碼一種鐵離子還原酶和銅離子還原酶。而與此相反,單倍體菌株?prk1的乙酸耐受性增強10mM,PRK1基因編碼一種蛋白質絲氨酸/蘇氨酸激酶。實驗表明結合SSR與SNP分子標記,可以利用較少的成本與信息鑒定出釀酒酵母乙酸耐受基因。并且本方法為研究酵母應對其他環境脅迫機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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q933

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本文編號：2305537