南极酵母Pseudozyma sp.JCC207海藻糖基因及其适应南极极端环境分子机制研究

发布时间：2018-02-03 22:19

  本文关键词： 南极酵母 Pseudozyma sp.JCC207 海藻糖 TPS1基因 TPS2基因 提取优化 功能分析　出处：《山东大学》2016年硕士论文　论文类型：学位论文

【摘要】：海藻糖是生物体中一种重要的非还原糖,对生物体组织和生物大分子具有非特异性保护作用,逐渐成为备受关注的天然物质。海藻糖具有在冻结、干燥、高渗透压等这类严酷的环境下,保护生物体膜、膜蛋白、DNA等的功效,使生物在极端环境中更容易存活,可见,海藻糖是生物体中重要的功能物质。南极酵母是指生活在南极海冰、海冰边缘或海水等极端环境中一大类酵母的总称,是南极主要的真菌生物。南极酵母长期在低温、高盐环境中生长和繁殖,海藻糖基因及海藻糖发挥了重要功能。南极酵母海藻糖合成酶指导合成海藻糖是其适应南极极端环境的重要生存机制,因此从分子水平上研究南极酵母的海藻糖产生机制是非常有必要的。南极酵母Pseudozyma sp. JCC207是南极酵母里具有代表性的一株菌,对它海藻糖形成机制研究可以为南极生境和其他物种的研究提供理论基础。本论文主要进行了以下研究内容：1)南极酵母Pseudozyma sp. JCC207的全基因组测序；2)普通PCR法克隆获得南极酵母Pseudozyma sp. JCC207基因rPSl并对TPS1、TPS2进行了生物信息学分析；3)应用实时定量PCR技术对南极酵母Pseudozyma sp. JCC207 TPS1的转录调控机制进行了研究；4)通过单因素实验法优化了南极酵母Pseudozyma sp. JCC207的培养条件；5)通过单因素实验、Box-Behnkens设计和响应面分析法对三氯乙酸法提取海藻糖的提取条件进行了优化,建立了影响因素的二次回归方程；6)通过添加不同浓度海藻糖与甘油、二甲基亚砜保种试剂对比,探究海藻糖对南极酵母Pseudozyma sp. JCC207保种的作用。全基因组测序得到了南极酵母Pseudozyma sp. JCC207全部基因,其中包含了本实验所需的TPS1、TPS2基因；克隆得到南极酵母Pseudozyma sp. JCC207 TPS1基因ORF全长1827 bp,编码蛋白608个氨基酸,预测蛋白相对分子质量为64.46 Da；测序得到TPS2基因ORF全长3963 bp,编码蛋白1321个氨基酸,预测蛋白相对分子质量为144.47 KDa; qRT-PCR结果显示,在高盐和低温条件下TPS1表达量高,说明,TPS1对高盐和低温敏感；单因素试验优化得到南极酵母最适培养温度为25℃,pH为5-9范围内酵母生长情况差别不大；响应面分析得到三氯乙酸法提取海藻糖的最佳条件为三氯乙酸浓度06 mol/L、体积15.16mL、提取时间2976 min,经验证,优化条件下的海藻糖提取量与理论预测值基本吻合；海藻糖保种实验中,功能试验表明,与甘油(20%)和二甲基亚砜(10%)等传统保种试剂相比,0.5%海藻糖溶液在短期内(5 d)对酵母具有更好的保种能力。
[Abstract]:Trehalose is an important non-reducing sugar in organism, which has nonspecific protective effect on organism tissue and biological macromolecule, and has gradually become a natural substance of much concern. Trehalose has been frozen and dried. Such harsh environments as high osmotic pressure protect the effectiveness of biological membranes, membrane proteins, DNA, and so on, so that organisms in extreme environments easier to survive, can be seen. Trehalose is an important functional substance in organisms. Antarctic yeast refers to a large category of yeast living in extreme environments such as Antarctic sea ice, sea ice edge or sea water. Antarctic yeast grows and reproduces in low temperature and high salt environment for a long time. Trehalose genes and trehalose play an important role in Antarctic yeast trehalose biosynthesis which is an important survival mechanism to adapt to Antarctic extreme environment. Therefore, it is necessary to study the mechanism of trehalose production in Antarctic yeast at molecular level. JCC207 is a representative strain of Antarctic yeast. The study on the mechanism of trehalose formation can provide a theoretical basis for the study of Antarctic habitats and other species. Genome sequencing of Antarctic yeast Pseudozyma sp. JCC207; 2) the Pseudozyma sp. JCC207 gene rPSl of Antarctic yeast was cloned by ordinary PCR method and the bioinformatics analysis of TPS1 and TPS2 was carried out. 3) the mechanism of transcriptional regulation of Pseudozyma sp. JCC207 TPS1 in Antarctic yeast was studied by real-time quantitative PCR. 4) the culture conditions of Antarctic yeast Pseudozyma sp. JCC207 were optimized by single factor experiment. 5) the extraction conditions of trehalose by trichloroacetic acid were optimized by Box-Behnkens design and response surface analysis, and the quadratic regression equation of influencing factors was established. 6) by adding different concentrations of trehalose and glycerol, dimethyl sulfoxide preservative reagent contrast. The effect of trehalose on species conservation of Antarctic yeast Pseudozyma sp. JCC207 was studied. All JCC207 genes. It contains the TPS1 / TPS2 gene needed in this experiment. The total length of ORF of Antarctic yeast Pseudozyma sp. JCC207 TPS1 was 1 827 BP, encoding 608 amino acids. The relative molecular weight of predicted protein was 64.46 Da. The total length of TPS2 gene ORF was 3963 BP, encoding 1321 amino acids, and the relative molecular weight of predicted protein was 144.47 KDa. The results of qRT-PCR showed that the expression of TPS1 was high under high salt and low temperature, indicating that TPS1 was sensitive to high salt and low temperature. The results of single factor experiment showed that the optimum culture temperature of Antarctic yeast was 25 鈩，

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