【摘要】：本實驗室前期從西藏Kefir粒中分離得到一株具有高產(chǎn)胞外多糖的馬奶酒樣乳桿菌ZW3,該菌具有良好的益生功能,在優(yōu)化的改良MRS培養(yǎng)基中,胞外多糖最高產(chǎn)量達2000mg/L以上,產(chǎn)生的胞外多糖具有較好的理化性質(zhì),是一株可用于食品,醫(yī)藥等多種領(lǐng)域的菌株。本論文以馬奶酒樣乳桿菌ZW3為研究對象,通過前期對其全基因組測序,對得到的結(jié)果進行分析,發(fā)現(xiàn)該菌株含有一個2.11 Mb的環(huán)狀染色體以及兩個大小分別為194,769 bp(pWW1)和46,296 bp (pWW2)的質(zhì)粒,還發(fā)現(xiàn)在馬奶酒樣乳桿菌ZW3染色體,有一段14.4 kb的胞外多糖基因簇,攜帶17個多糖合成相關(guān)基因,及許多胞外多糖合成通路中的編碼胞外多糖代謝通路中代謝關(guān)鍵酶的管家基因pgm2, galE, galK等。為了研究馬奶酒樣乳桿菌ZW3的代謝通路,尋找高效胞外多糖產(chǎn)糖通路,以及構(gòu)建過表達菌株高產(chǎn)胞外多糖,以馬奶酒樣乳桿菌ZW3染色體為模板,利用設(shè)計的引物對代謝管家基因pgm2進行了擴增,構(gòu)建大腸桿菌重組質(zhì)粒pET30a-pgm2,轉(zhuǎn)化至大腸桿菌中,并成功進行了表達。同時,構(gòu)建構(gòu)建了乳酸菌重組質(zhì)粒pMG36e-pgm2,轉(zhuǎn)化至馬奶酒樣乳桿菌ZW3菌株中,成功構(gòu)建了過表達菌株,并對馬奶酒樣乳桿菌ZW3野生菌以及其轉(zhuǎn)化菌株進行生長特性分析。針對已經(jīng)轉(zhuǎn)化成功的重組菌株,以野生菌馬奶酒樣乳桿菌ZW3為對照,利用苯酚-硫酸法檢測兩者的胞外多糖產(chǎn)量,結(jié)果顯示胞外多糖產(chǎn)量提高了20.16%。根據(jù)SDS-PAGE電泳圖譜可初步確定α-磷酸葡萄糖變位酶基pgm2在大腸桿菌以及馬奶酒樣乳桿菌ZW3中成功表達了蛋白。通過本實驗的結(jié)果分析和參考文獻報道,認為α-磷酸葡萄糖變位酶基因pgm2在胞外多糖的合成代謝通路中發(fā)揮了重要的作用。
[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 偽 -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 偽 -phosphate glucose translocation enzyme gene pgm2 plays an important role in the biosynthesis and metabolism of extracellular polysaccharides.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q93;Q78

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1 崔月倩;α-磷酸葡萄糖變位酶基因pgm2的克隆表達及功能研究[D];天津科技大學(xué);2015年

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本文編號：2234061