α-磷酸葡萄糖变位酶基因pgm2的克隆表达及功能研究
发布时间：2018-09-10 09:33
【摘要】：本实验室前期从西藏Kefir粒中分离得到一株具有高产胞外多糖的马奶酒样乳杆菌ZW3,该菌具有良好的益生功能,在优化的改良MRS培养基中,胞外多糖最高产量达2000mg/L以上,产生的胞外多糖具有较好的理化性质,是一株可用于食品,医药等多种领域的菌株。本论文以马奶酒样乳杆菌ZW3为研究对象,通过前期对其全基因组测序,对得到的结果进行分析,发现该菌株含有一个2.11 Mb的环状染色体以及两个大小分别为194,769 bp(pWW1)和46,296 bp (pWW2)的质粒,还发现在马奶酒样乳杆菌ZW3染色体,有一段14.4 kb的胞外多糖基因簇,携带17个多糖合成相关基因,及许多胞外多糖合成通路中的编码胞外多糖代谢通路中代谢关键酶的管家基因pgm2, galE, galK等。为了研究马奶酒样乳杆菌ZW3的代谢通路,寻找高效胞外多糖产糖通路,以及构建过表达菌株高产胞外多糖,以马奶酒样乳杆菌ZW3染色体为模板,利用设计的引物对代谢管家基因pgm2进行了扩增,构建大肠杆菌重组质粒pET30a-pgm2,转化至大肠杆菌中,并成功进行了表达。同时,构建构建了乳酸菌重组质粒pMG36e-pgm2,转化至马奶酒样乳杆菌ZW3菌株中,成功构建了过表达菌株,并对马奶酒样乳杆菌ZW3野生菌以及其转化菌株进行生长特性分析。针对已经转化成功的重组菌株,以野生菌马奶酒样乳杆菌ZW3为对照,利用苯酚-硫酸法检测两者的胞外多糖产量,结果显示胞外多糖产量提高了20.16%。根据SDS-PAGE电泳图谱可初步确定α-磷酸葡萄糖变位酶基pgm2在大肠杆菌以及马奶酒样乳杆菌ZW3中成功表达了蛋白。通过本实验的结果分析和参考文献报道,认为α-磷酸葡萄糖变位酶基因pgm2在胞外多糖的合成代谢通路中发挥了重要的作用。
[Abstract]:A strain of Lactobacillus equi with high exopolysaccharide (EPS) was isolated from Tibetan Kefir grains in our laboratory. The strain has good probiotic function. In the optimized modified MRS medium, the highest yield of extracellular polysaccharides was over 2000mg/L. The extracellular polysaccharides produced have good physicochemical properties and can be used in many fields, such as food, medicine and so on. In this paper, the whole genome of Lactobacillus equina (ZW3) was sequenced and the results were analyzed. It was found that the strain contained a circular chromosome of 2.11 Mb and two plasmids of 194769 bp (pWW1) and 46296 bp (pWW2), and that there was an exopolysaccharide gene cluster of 14.4 kb on ZW3 chromosome of Lactobacillus equina. There are 17 genes associated with polysaccharide synthesis, and a housekeeping gene, pgm2, galE, galK, which encodes the key enzymes in the metabolism of extracellular polysaccharides in many extracellular polysaccharide biosynthesis pathways. In order to study the metabolic pathway of Lactobacillus equina (ZW3), to search for high efficient extracellular polysaccharide (EPS) production pathway, and to construct the overexpression of EPS, the ZW3 chromosome of Lactobacillus equina was used as a template. The metabolic housekeeper gene pgm2 was amplified by using the designed primers. The recombinant plasmid pET30a-pgm2, was constructed and transformed into E. coli and successfully expressed. At the same time, the recombinant plasmid pMG36e-pgm2, of Lactobacillus lactobacillus was constructed and transformed into ZW3 strain of Lactobacillus lactobacillus. The overexpression strain was successfully constructed, and the growth characteristics of ZW3 wild strain and its transformed strain were analyzed. The yield of extracellular polysaccharides was detected by phenol-sulfuric acid method with ZW3 of Lactobacillus equi. The results showed that the yield of extracellular polysaccharides was increased by 20.16%. According to SDS-PAGE electrophoretic patterns, it was preliminarily confirmed that 伪 -phosphate glucose translocation enzyme pgm2 was successfully expressed in Escherichia coli and Lactobacillus equi ZW3. Through the analysis of the results of this experiment and the references, it is concluded that the 伪 -phosphate glucose translocation enzyme gene pgm2 plays an important role in the biosynthesis and metabolism of extracellular polysaccharides.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q93;Q78
本文编号：2234061
[Abstract]:A strain of Lactobacillus equi with high exopolysaccharide (EPS) was isolated from Tibetan Kefir grains in our laboratory. The strain has good probiotic function. In the optimized modified MRS medium, the highest yield of extracellular polysaccharides was over 2000mg/L. The extracellular polysaccharides produced have good physicochemical properties and can be used in many fields, such as food, medicine and so on. In this paper, the whole genome of Lactobacillus equina (ZW3) was sequenced and the results were analyzed. It was found that the strain contained a circular chromosome of 2.11 Mb and two plasmids of 194769 bp (pWW1) and 46296 bp (pWW2), and that there was an exopolysaccharide gene cluster of 14.4 kb on ZW3 chromosome of Lactobacillus equina. There are 17 genes associated with polysaccharide synthesis, and a housekeeping gene, pgm2, galE, galK, which encodes the key enzymes in the metabolism of extracellular polysaccharides in many extracellular polysaccharide biosynthesis pathways. In order to study the metabolic pathway of Lactobacillus equina (ZW3), to search for high efficient extracellular polysaccharide (EPS) production pathway, and to construct the overexpression of EPS, the ZW3 chromosome of Lactobacillus equina was used as a template. The metabolic housekeeper gene pgm2 was amplified by using the designed primers. The recombinant plasmid pET30a-pgm2, was constructed and transformed into E. coli and successfully expressed. At the same time, the recombinant plasmid pMG36e-pgm2, of Lactobacillus lactobacillus was constructed and transformed into ZW3 strain of Lactobacillus lactobacillus. The overexpression strain was successfully constructed, and the growth characteristics of ZW3 wild strain and its transformed strain were analyzed. The yield of extracellular polysaccharides was detected by phenol-sulfuric acid method with ZW3 of Lactobacillus equi. The results showed that the yield of extracellular polysaccharides was increased by 20.16%. According to SDS-PAGE electrophoretic patterns, it was preliminarily confirmed that 伪 -phosphate glucose translocation enzyme pgm2 was successfully expressed in Escherichia coli and Lactobacillus equi ZW3. Through the analysis of the results of this experiment and the references, it is concluded that the 伪 -phosphate glucose translocation enzyme gene pgm2 plays an important role in the biosynthesis and metabolism of extracellular polysaccharides.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q93;Q78
【相似文献】
相关硕士学位论文 前1条
1 崔月倩;α-磷酸葡萄糖变位酶基因pgm2的克隆表达及功能研究[D];天津科技大学;2015年
，本文编号：2234061
本文链接：https://www.wllwen.com/shoufeilunwen/benkebiyelunwen/2234061.html
