【摘要】：黃酒是我國的傳統(tǒng)釀造食品,在生產(chǎn)黃酒的過程中,釀酒酵母(Saccharomyces cerevisiae)作為關(guān)鍵微生物對(duì)酒精發(fā)酵起主導(dǎo)作用,即配合紅曲和麥曲等發(fā)揮共同作用將糖轉(zhuǎn)化成酒,所得黃酒具有獨(dú)特的風(fēng)味、豐富的營養(yǎng)物質(zhì)及較高的保健功效。目前,黃酒酵母不僅在傳統(tǒng)的黃酒發(fā)酵中得到廣泛應(yīng)用,而且在機(jī)械的黃酒大生產(chǎn)中也取得了良好的社會(huì)效益和經(jīng)濟(jì)效益。在黃酒生產(chǎn)中,發(fā)酵環(huán)境發(fā)生了變化比如溫度、滲透壓、pH、乙醇濃度等,這些變化都會(huì)對(duì)酵母的發(fā)酵生長產(chǎn)生比較明顯的影響。乙醇是制約酒精生產(chǎn)的主要因素之一。本文對(duì)本實(shí)驗(yàn)室保存的從黃酒發(fā)酵工廠分離獲得的黃酒酵母作為對(duì)象,對(duì)其進(jìn)行進(jìn)一步分離篩選,最終分離獲得一酵母單倍體 YHJ7(D2)(Saccharomyces cerevisiae),它具有高耐受性的良好特質(zhì)。通過對(duì)這株具有優(yōu)良抗逆性的黃酒酵母菌進(jìn)行全基因組測序、組裝并通過比較基因組學(xué)及生物信息學(xué)分析該菌的特異性。在與同樣黃酒發(fā)酵工廠獲得的釀酒酵母N85(Saccharomycescerevisiae)基因組比較分析的時(shí)候發(fā)現(xiàn)了一些可能差異片段,且在與模式實(shí)驗(yàn)菌株S288c(Saccharomyces cerevisiae)進(jìn)行比較分析時(shí),發(fā)現(xiàn)了一些不在實(shí)驗(yàn)菌株S288c存在,卻在該黃酒酵母菌中存在的新基因,例如g5170。本文在完成基因組組裝及分析的基礎(chǔ)上主要對(duì)新基因g5170做了功能性研究與驗(yàn)證。通過生物信息學(xué)分析,本實(shí)驗(yàn)室預(yù)測新基因g5170的功能可能與乙醇耐受性有很大關(guān)系。為了進(jìn)一步確認(rèn)新基因g5170與乙醇耐受性有關(guān),本實(shí)驗(yàn)室通過敲除與過表達(dá)g5170基因兩方面研究與驗(yàn)證它的功能。本實(shí)驗(yàn)室前期利用Cre/loxP系統(tǒng)敲除目的基因g5170構(gòu)建此基因的敲除菌株,并對(duì)基因敲除菌株與原始菌株進(jìn)行耐受性比較分析。結(jié)果表明敲除菌株與原始菌株對(duì)高糖和高溫的耐受性上的表現(xiàn)近乎相同,但是對(duì)于高濃度乙醇環(huán)境脅迫下,基因敲除菌株YHJ7-Δg5170的乙醇耐受性要比原始菌株YHJ7表現(xiàn)的弱很多。為進(jìn)一步驗(yàn)證新基因g5170與乙醇耐受性的關(guān)系,本實(shí)驗(yàn)利用釀酒酵母表達(dá)載體質(zhì)粒pYES2 將新基因 g5170 轉(zhuǎn)化到 BY4741(Saccharomyces cerevisiae)酵母菌株中,利用半乳糖誘導(dǎo)法誘導(dǎo)新基因在BY4741重組菌中表達(dá),數(shù)據(jù)顯示g5170的重組酵母菌株對(duì)乙醇耐受性明顯比對(duì)照菌株強(qiáng)。綜上結(jié)果表明此基因的功能確實(shí)與黃酒酵母菌適應(yīng)乙醇脅迫有關(guān)。
[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.
【學(xué)位授予單位】：浙江工商大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS261.11

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