酿酒酵母NDE1基因缺失突变株的构建及其生物学性质研究

发布时间：2018-05-17 21:13

本文选题：酿酒酵母 + NDE1基因　；参考：《南京农业大学》2013年硕士论文

【摘要】：奶牛于产前2-3周至产后2-3周处于围产期。这一时期对奶牛的健康和生产性能影响极大,以能量摄入减少和能量需求增加所导致的能量负平衡为特点。奶牛酮病和脂肪肝(妊娠毒血症、肥胖综合征)是以围产期能量代谢障碍为病理学基础的常见群发疾病。甘油具有生糖作用,进入糖异生途径后不依赖限速酶的催化,可以有效缓解奶牛围产期能量负平衡。发酵法生产甘油因其污染低毒性小而受到广泛关注。本实验室拥有自主知识产权的产甘油酿酒酵母NAU-ZH-GY1是农业部公布的允许作为饲用的益生菌,对其进行基因改造后使其甘油产量提高,将工程菌发酵液制成饲料添加剂可以同时发挥甘油和益生菌的双重作用。本试验从酵母代谢原理出发,构建NDE1基因缺失突变菌株,部分切断线粒体外NADH的脱氢反应,胞质中NADH向线粒体呼吸链的传递受到影响,最终有更多的NADH进入甘油合成通路使得甘油产量升高,为研发一种防控围产期奶牛能量代谢障碍性疾病的微生态制剂提供条件。试验1酿酒酵母NDEl缺失突变株的构建本试验以本实验室拥有自主知识产权的酿酒酵母NAU-ZH-GY1为出发菌株,利用Cre/loxP重组系统成功缺失NDEl基因一条等位基因,在原基因位置留下一个loxP位点,降低了外来基因的生物危害,与传统敲除方法相比更加准确、简便和高效。针对酿酒酵母NAU-ZH-GY1优化出最佳基因缺失电转方案,即使用]DTTBuffet(DTT 20mlM)预处理处于对数生长中期的酵母细胞30 min,外源DNA浓度控制在400 ng·μL-1,电压3 kV,电容25μF,电阻200 Ω。之后利用酵母等位基因分离及融合技术,成功构建ndel△完全缺失突变菌株NAU-ZH-GY1-ndel、△,此菌株不含任何抗性标记及大段外源无意义基因,符合相关规定对益生菌安全性的要求。工程菌构建过程采用的方法成本低廉效率高,节约了大量时间及科研经费。试验2酿酒酵母NDE1缺失突变株生物学性质研究本试验对试验1构建的酿酒酵母NAU-ZH-GY1-ndel△和出发菌株NAU-ZH-GY1进行生物学性质研究,分别对菌株的生长状况、稳定性、甘油产量、生物量和发酵液中活菌数进行测定。摇瓶发酵试验结果表明两个菌株在生长状况和活菌数方面无显著差异,酿酒酵母NAU-ZH-GY1-ndel△遗传性质稳定。受基因缺失影响,酿酒酵母NAU-ZH-GY1-ndel△生物量与出发菌株相比下降了17.1%,差异显著(p0.05)。酿酒酵母NAU-ZH-GY1-ndel△甘油产量为26.61 g.L-1,比出发菌株提高了12.9%,与出发菌株组相比差异显著(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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：Q93

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