黄酒酵母菌株YHJ7的基因组分析及基因功能研究

发布时间：2018-10-13 16:51

【摘要】：黄酒是我国的传统酿造食品,在生产黄酒的过程中,酿酒酵母(Saccharomyces cerevisiae)作为关键微生物对酒精发酵起主导作用,即配合红曲和麦曲等发挥共同作用将糖转化成酒,所得黄酒具有独特的风味、丰富的营养物质及较高的保健功效。目前,黄酒酵母不仅在传统的黄酒发酵中得到广泛应用,而且在机械的黄酒大生产中也取得了良好的社会效益和经济效益。在黄酒生产中,发酵环境发生了变化比如温度、渗透压、pH、乙醇浓度等,这些变化都会对酵母的发酵生长产生比较明显的影响。乙醇是制约酒精生产的主要因素之一。本文对本实验室保存的从黄酒发酵工厂分离获得的黄酒酵母作为对象,对其进行进一步分离筛选,最终分离获得一酵母单倍体 YHJ7(D2)(Saccharomyces cerevisiae),它具有高耐受性的良好特质。通过对这株具有优良抗逆性的黄酒酵母菌进行全基因组测序、组装并通过比较基因组学及生物信息学分析该菌的特异性。在与同样黄酒发酵工厂获得的酿酒酵母N85(Saccharomycescerevisiae)基因组比较分析的时候发现了一些可能差异片段,且在与模式实验菌株S288c(Saccharomyces cerevisiae)进行比较分析时,发现了一些不在实验菌株S288c存在,却在该黄酒酵母菌中存在的新基因,例如g5170。本文在完成基因组组装及分析的基础上主要对新基因g5170做了功能性研究与验证。通过生物信息学分析,本实验室预测新基因g5170的功能可能与乙醇耐受性有很大关系。为了进一步确认新基因g5170与乙醇耐受性有关,本实验室通过敲除与过表达g5170基因两方面研究与验证它的功能。本实验室前期利用Cre/loxP系统敲除目的基因g5170构建此基因的敲除菌株,并对基因敲除菌株与原始菌株进行耐受性比较分析。结果表明敲除菌株与原始菌株对高糖和高温的耐受性上的表现近乎相同,但是对于高浓度乙醇环境胁迫下,基因敲除菌株YHJ7-Δg5170的乙醇耐受性要比原始菌株YHJ7表现的弱很多。为进一步验证新基因g5170与乙醇耐受性的关系,本实验利用酿酒酵母表达载体质粒pYES2 将新基因 g5170 转化到 BY4741(Saccharomyces cerevisiae)酵母菌株中,利用半乳糖诱导法诱导新基因在BY4741重组菌中表达,数据显示g5170的重组酵母菌株对乙醇耐受性明显比对照菌株强。综上结果表明此基因的功能确实与黄酒酵母菌适应乙醇胁迫有关。
[Abstract]:Yellow rice wine is the traditional brewing food in our country. In the process of producing rice wine, Saccharomyces cerevisiae (Saccharomyces cerevisiae), as a key microorganism, plays a leading role in alcohol fermentation, that is, combining with red koji and wheat koji, sugar can be converted into wine. The obtained yellow rice wine has unique flavor, rich nutrition and high health care effect. At present, rice wine yeast is not only widely used in the traditional rice wine fermentation, but also has achieved good social and economic benefits in the large-scale production of mechanical rice wine. In the production of rice wine, the fermentation environment has changed, such as temperature, osmotic pressure, pH, ethanol concentration and so on, these changes will have a more obvious impact on the fermentation growth of yeast. Ethanol is one of the main factors restricting alcohol production. In this paper, the rice wine yeast isolated from the rice wine fermentation factory was isolated and screened in our laboratory, and a yeast haploid YHJ7 (D2) (Saccharomyces cerevisiae),) was obtained with high tolerance. The whole genome of this strain was sequenced and its specificity was analyzed by comparative genomics and bioinformatics. Some possible differential fragments were found in the genome analysis of Saccharomyces cerevisiae N85 (Saccharomycescerevisiae) obtained from the same rice wine fermentation plant, and some were found not in the experimental strain S288c when compared with the model strain S288c (Saccharomyces cerevisiae). New genes, such as g5170. 0. On the basis of complete genome assembly and analysis, the functional study and validation of the new gene g5170 were carried out in this paper. By bioinformatics analysis, we predicted that the function of the new gene g5170 might be related to ethanol tolerance. In order to further confirm the relationship between the new gene g5170 and ethanol tolerance, the function of the novel gene g5170 was studied and verified by knockout and overexpression of G5170 gene in our laboratory. In our laboratory, we used Cre/loxP system to knockout the target gene g5170 and constructed the knockout strain, and compared the tolerance between the gene knockout strain and the original strain. The results showed that the tolerance of knockout strain to high sugar and high temperature was almost the same as that of original strain, but the ethanol tolerance of gene knockout strain YHJ7- 螖 g5170 was much weaker than that of original strain YHJ7 under high ethanol stress. In order to further verify the relationship between the expression of new gene g5170 and ethanol tolerance, the new gene g5170 was transformed into BY4741 (Saccharomyces cerevisiae) yeast strain by recombinant plasmid pYES2 of Saccharomyces cerevisiae. Galactose induction was used to induce the expression of the new gene in BY4741 recombinant strain. The data showed that the recombinant yeast strain g5170 had stronger tolerance to ethanol than the control strain. The results showed that the function of the gene was related to the adaptation of yeasts to ethanol stress.
【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS261.11

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