本文選題：釀酒酵母 + NDE1基因��； 參考：《南京農(nóng)業(yè)大學(xué)》2013年碩士論文

【摘要】：奶牛于產(chǎn)前2-3周至產(chǎn)后2-3周處于圍產(chǎn)期。這一時期對奶牛的健康和生產(chǎn)性能影響極大,以能量攝入減少和能量需求增加所導(dǎo)致的能量負(fù)平衡為特點。奶牛酮病和脂肪肝(妊娠毒血癥、肥胖綜合征)是以圍產(chǎn)期能量代謝障礙為病理學(xué)基礎(chǔ)的常見群發(fā)疾病。甘油具有生糖作用,進(jìn)入糖異生途徑后不依賴限速酶的催化,可以有效緩解奶牛圍產(chǎn)期能量負(fù)平衡。發(fā)酵法生產(chǎn)甘油因其污染低毒性小而受到廣泛關(guān)注。本實驗室擁有自主知識產(chǎn)權(quán)的產(chǎn)甘油釀酒酵母NAU-ZH-GY1是農(nóng)業(yè)部公布的允許作為飼用的益生菌,對其進(jìn)行基因改造后使其甘油產(chǎn)量提高,將工程菌發(fā)酵液制成飼料添加劑可以同時發(fā)揮甘油和益生菌的雙重作用。本試驗從酵母代謝原理出發(fā),構(gòu)建NDE1基因缺失突變菌株,部分切斷線粒體外NADH的脫氫反應(yīng),胞質(zhì)中NADH向線粒體呼吸鏈的傳遞受到影響,最終有更多的NADH進(jìn)入甘油合成通路使得甘油產(chǎn)量升高,為研發(fā)一種防控圍產(chǎn)期奶牛能量代謝障礙性疾病的微生態(tài)制劑提供條件。試驗1釀酒酵母NDEl缺失突變株的構(gòu)建本試驗以本實驗室擁有自主知識產(chǎn)權(quán)的釀酒酵母NAU-ZH-GY1為出發(fā)菌株,利用Cre/loxP重組系統(tǒng)成功缺失NDEl基因一條等位基因,在原基因位置留下一個loxP位點,降低了外來基因的生物危害,與傳統(tǒng)敲除方法相比更加準(zhǔn)確、簡便和高效。針對釀酒酵母NAU-ZH-GY1優(yōu)化出最佳基因缺失電轉(zhuǎn)方案,即使用]DTTBuffet(DTT 20mlM)預(yù)處理處于對數(shù)生長中期的酵母細(xì)胞30 min,外源DNA濃度控制在400 ng·μL-1,電壓3 kV,電容25μF,電阻200 Ω。之后利用酵母等位基因分離及融合技術(shù),成功構(gòu)建ndel△完全缺失突變菌株NAU-ZH-GY1-ndel、△,此菌株不含任何抗性標(biāo)記及大段外源無意義基因,符合相關(guān)規(guī)定對益生菌安全性的要求。工程菌構(gòu)建過程采用的方法成本低廉效率高,節(jié)約了大量時間及科研經(jīng)費。試驗2釀酒酵母NDE1缺失突變株生物學(xué)性質(zhì)研究本試驗對試驗1構(gòu)建的釀酒酵母NAU-ZH-GY1-ndel△和出發(fā)菌株NAU-ZH-GY1進(jìn)行生物學(xué)性質(zhì)研究,分別對菌株的生長狀況、穩(wěn)定性、甘油產(chǎn)量、生物量和發(fā)酵液中活菌數(shù)進(jìn)行測定。搖瓶發(fā)酵試驗結(jié)果表明兩個菌株在生長狀況和活菌數(shù)方面無顯著差異,釀酒酵母NAU-ZH-GY1-ndel△遺傳性質(zhì)穩(wěn)定。受基因缺失影響,釀酒酵母NAU-ZH-GY1-ndel△生物量與出發(fā)菌株相比下降了17.1%,差異顯著(p0.05)。 釀酒酵母NAU-ZH-GY1-ndel△甘油產(chǎn)量為26.61 g.L-1,比出發(fā)菌株提高了12.9%,與出發(fā)菌株組相比差異顯著(p0.05)。
[Abstract]:The cows were in perinatal period from 2-3 weeks to 2-3 weeks postpartum. This period has a great impact on the health and production performance of dairy cows, which is characterized by the negative energy balance caused by the decrease of energy intake and the increase of energy demand. Ketosis and fatty liver (gestational toxemia, obesity syndrome) are common diseases based on perinatal energy metabolism disorders. Glycerol has the effect of sugar generation, and it can alleviate the negative energy balance in the perinatal period of dairy cows without the catalysis of rate-limiting enzyme after entering the glucose allogeneic pathway. The production of glycerol by fermentation has attracted wide attention because of its low pollution and low toxicity. NAU-ZH-GY1, which has its own intellectual property rights, is a probiotic bacterium approved by the Ministry of Agriculture for forage, which has been genetically modified to increase its glycerol production. The double effects of glycerol and probiotics can be played by making the fermentation broth of engineering bacteria into feed additive. Based on the principle of yeast metabolism, a mutant strain with deletion of NDE1 gene was constructed, which partially cut off the dehydrogen reaction of NADH outside mitochondria and affected the transmission of NADH from cytoplasm to respiratory chain of mitochondria. Finally, more NADH enter the glycerol synthesis pathway to increase glycerol production, which provides conditions for the development of a microecological preparation for the prevention and control of perinatal energy metabolic disorders in dairy cows. Construction of NDEl deletion Mutant of Saccharomyces cerevisiae in experiment 1 A allele of NDEl gene was successfully deleted by Cre/loxP recombination system based on NAU-ZH-GY1 of Saccharomyces cerevisiae which has its own intellectual property rights in our laboratory. Leaving a loxP site in the original gene position reduces the biological damage of foreign genes and is more accurate, simple and efficient than traditional knockout methods. According to the optimal electroporation protocol of gene deletion in Saccharomyces cerevisiae (NAU-ZH-GY1), the optimal gene deletion electroporation scheme was optimized, that is, the yeast cells in logarithmic growth period were pretreated with] DTTBuffet(DTT 20ml M) for 30 min, the concentration of exogenous DNA was controlled at 400ng 渭 L -1, the voltage was 3 kV, the capacitance was 25 渭 F, and the resistance was 200 惟. Then the yeast allele isolation and fusion technique were used to construct the completely deleted ndel mutant NAU-ZH-GY1-ndel. the strain did not contain any resistance markers and large segments of exogenous meaningless genes, which met the requirements of the safety of probiotics. The method used in the construction process of engineering bacteria is low cost and high efficiency, and saves a lot of time and research expenses. Study on Biological Properties of Saccharomyces cerevisiae NDE1 deletion Mutant in experiment 2. The biological properties of NAU-ZH-GY1-ndel and NAU-ZH-GY1 were studied in this experiment. The growth status, stability and glycerol yield of the strain were studied respectively. Biomass and the number of live bacteria in fermentation broth were determined. The results of shaking flask fermentation showed that there was no significant difference in growth status and number of live bacteria between the two strains, and the genetic properties of Saccharomyces cerevisiae NAU-ZH-GY1-ndel were stable. Under the influence of gene deletion, the NAU-ZH-GY1-ndel biomass of Saccharomyces cerevisiae decreased by 17.1% compared with the original strain, and the difference was significant (p 0.05). The glycerol yield of Saccharomyces cerevisiae NAU-ZH-GY1-ndel was 26.61 g 路L ~ (-1), which was 12.9g 路L ~ (-1) higher than that of the original strain.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：Q93

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