导　　师： 朱咏华

学科专业： G1010

授予学位： 硕士

作　　者： ;

机构地区： 湖南大学

摘　　要： 化石能源是当今能源结构的主体,却是一类非常宝贵的不可再生资源。面对化石能源日益短缺及其燃烧产生的环境污染问题日益严重的现状,人们开始寻找清洁可持续利用的替代能源。因此,开发和利用生物质能源己成为一种战略选择。生物质能源产业中,目前规模方面发展最快的是燃料乙醇,但成本高昂是制约微生物发酵生产乙醇工业的瓶颈。尤其是对于同步糖化发酵法生产乙醇,糖化和发酵时的最适温度不协调以及乙醇的抑制作用是急待解决的问题。研究开发出适用于同步糖化发酵技术所需高耐受性酵母菌株,就可以提高燃料乙醇的生产效率、降低成本。因此,本论文针对与酵母中能耐受高浓度糖及高浓度乙醇的基因——转录因子SPT15进行基因工程改造,期望能获得高耐受性酵母菌株。对SPT15进行了不同长度片段的随机突变,并将产物连接到含有半乳糖启动子的酵母穿梭载体质粒PYES2(多拷贝质粒)和PGAL1-YCPLAC22(单拷贝质粒)中,使突变基因能被诱导过量表达。随机突变采用在标准PCR反应体系中加入MN2+的方式,通过对不同片段长度的SPT15基因随机突变的体系进行的系统研究,我们发现大于500BP长度的DNA随着MN2+浓度的提高,突变率迅速上升,而小于250 BP短片段的DNA对MN2+浓度不特别敏感,从而进一步确定了对1000 BP长度以内不同长度片断单突变或多突变所适合的MN2+浓度。同时,在建立突变体库的过程中,对大肠杆菌电转化条件进行了摸索,确定了电转化的最佳方式。本文构建的SPT15突变体库为进一步筛选高耐受酵母菌创造了条件,为酵母基因工程改造提供了有价值的实验经验。 Fossil fuels is the subject of today's energy structure, and it is a very valuable non-renewable resources. Faced with a growing shortage of fossil fuels, as well as its increasingly serious environmental pollution problems, it forces people to start looking for the sustainable use of cleaner alternative energy sources. Therefore, the development and utilization of biomass energy has become a strategic choice. At present, the scale of fuel ethanol is the fastest-growing in biomass energy industry, but the high cost of microbial fermentation for ethanol production is the bottlenecks in industry conditions. In order to research for yeast strains of high tolerance for ethanol production of simultaneous saccharification and fermentation technology to increase fuel ethanol production efficiency and reduce the costs, in this paper, with the yeast tolerance for high concentrations of sugar and ethanol gene——transcription factor SPT15 genetically engineered to carry out. The products that are random and site-directed mutagenesis of the gene SPT15 are connected to the plasmids PYES2 and Ycplac22, and over-express genes. The plasmids are the shuttle vector plasmid between E.coil and yeast and contain GALⅠpromoter,but they don’t have the galactose genes. Adding Mn~/(2+/) in the standard PCR reaction system can reduce effectively the fidelity of Taq DNA polymerase, and cause random mutagenesis. The DNA fragments of different lengths are differently effective on the concentration of Mn~/(2+/). By exploring that effects, we can conclude that more than 500bp DNA has a rapid increase in mutation rate with the Mn~/(2+/) concentration improvement, but less than 250bp DNA is not particularly sensitive on the Mn~/(2+/) concentration. At the same time,when mutant library was established, we explored different transformation methods to determine the best conditions for electroporation. The mutant library of SPT15 creates the conditions for screening of high-tolerance yeast and provide valuable experimental experience.

关 键 词： 燃料乙醇 同步糖化发酵 酵母菌株 基因改造 突变体库

分 类 号： [TQ517.43 TQ920.1]

领　　域： [化学工程] [轻工技术与工程]