布拉氏酵母菌细胞壁高含量β-葡聚糖菌株的构建及功能的研究

发布时间：2018-01-21 08:16

本文关键词： 布拉氏酵母菌 β-葡聚糖基因敲除益生功能基因工程　出处：《山东农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：布拉氏酵母菌(S.boulardii)是法国科学家Henri Boulard于1920年从印尼荔枝等水果中分离得到的一种益生酵母菌。目前,布拉菌作为微生态制剂在欧美等国家广泛用于防治旅行者和儿童的各种腹泻。畜牧业上,在饲料中添加布拉氏酵母菌制剂以提高动物的生产性能来提升经济效益已得到包括中国、欧盟在内的世界上许多国家的认可。随着人们对健康和生态环境的高度重视及在畜禽养殖中的进一步研究,布拉菌在畜牧业和医学上必将发挥更大的作用,同时也会有更广阔的应用前景。布拉氏酵母菌作为最具有发展潜力的益生菌,其细胞壁的主要组成成分β-葡聚糖在布拉菌作为益生菌整体的作用中起着关键作用。酵母菌β-葡聚糖是一种具有增强免疫力活性的多糖,占布拉氏酵母菌细胞壁组成的50%以上,因此,布拉菌无疑是β-葡聚糖的重要来源之一。然而,对于β-葡聚糖功能的研究,大都是针对分离纯化的β-葡聚糖进行的。β-葡聚糖作为布拉菌细胞壁的主要组成成分,在布拉菌作为益生菌整体的作用中,不同β-葡聚糖含量与布拉菌的益生功能的影响却未见研究和报道。因此,本研究主要是通过Cre-loxP基因敲除系统对目标基因进行基因敲除,构建一个布拉菌细胞壁高含量β-葡聚糖的变异菌株,可丰富β-葡聚糖的自然资源,为工业上大量提取β-葡聚糖提供了原材料。同时,研究敲除目标基因后对布拉菌表型的影响,并通过体外及动物实验研究β-葡聚糖在布拉菌作为益生菌的益生功能中的作用,以及布拉菌细胞壁β-葡聚糖含量的变化对其益生功能、非特异性免疫能力、抗感染能力及生产性能的影响,探讨布拉菌在分子水平上的作用机理,为进一步改善其益生特性打下基础和提供技术支持。本研究根据研究的目的和意义选出1个可明显影响布拉氏酵母菌细胞壁β-葡聚糖含量的基因THP1,通过实验室构建的适合布拉菌的Cre-loxP基因敲除系统成功筛选出布拉菌细胞壁高含量β-葡聚糖的变异菌株Sb-thp1。对杂合子菌株、变异株的表型进行研究发现,和野生型布拉菌相比,变异株的生长速度减慢,细胞形态变圆变大,而杂合子菌株无明显变化;杂合子菌株、变异株细胞壁对刚果红、NaCl、SDS的敏感性无明显变化,但细胞壁β-葡聚糖的含量明显升高,这丰富了β-葡聚糖的自然资源。动物实验结果表明,布拉菌细胞壁高含量β-葡聚糖变异株和野生型布拉菌相比没有显著提高试验动物的生产性能,其增重效果不明显;对试验动物免疫器官的生长没有明显的促进作用,但变异株组的试验动物的脾脏指数最高;布拉菌THP1缺失变异株有一定的抗大肠杆菌感染的能力,能降低试验动物的死亡率。
[Abstract]:S. boulardii is a probiotic yeast isolated from litchi and other fruits of Indonesia in 1920 by French scientist Henri Boulard. As a microecological agent, Brassichia coli is widely used to prevent and cure diarrhea and animal husbandry of travelers and children in Europe and the United States and other countries. The addition of Saccharomyces brassicus to feed to improve animal productivity to improve economic benefits has been achieved in China. With the attention paid to health and ecological environment and further research in livestock and poultry breeding, Brassicus will play a more important role in animal husbandry and medicine. At the same time, it will also have a wider application prospect. As the most potential probiotics, Saccharomyces brasiliformis is the most promising. 尾 -glucan, a major component of the cell wall, plays a key role in the role of Brassicus as a probiotic organism, and yeast 尾 -glucan is a polysaccharide with enhanced immunity. More than 50% of the cell wall of Saccharomyces cerevisiae is composed of 尾 -glucan. However, the function of 尾 -glucan is studied. 尾 -glucan is the main component of the cell wall of Brassicus, which acts as a probiotic. The effects of different 尾 -glucan content on the prebiotic function of Brassicus have not been studied and reported. Therefore, the target gene was knockout by Cre-loxP gene knockout system. The construction of a mutant strain with high content of 尾 -glucan in cell wall of Brassicus can enrich the natural resources of 尾 -glucan and provide raw materials for industrial extraction of 尾 -glucan. To study the effect of knockout on the phenotype of Brassicus, and to study the role of 尾 -glucan in the probiotic function of Brassicus as probiotics through in vitro and animal experiments. The effects of 尾 -glucan content on probiotic function, non-specific immunity, anti-infection ability and production performance were also discussed. In order to further improve its probiotic characteristics and provide technical support, according to the purpose and significance of the study, we selected a gene THP1 that can significantly affect the content of 尾 -glucan in the cell wall of Saccharomyces brasiliensis. The mutant strain Sb-thp1 with high content of 尾 -glucan in cell wall was successfully screened by the Cre-loxP gene knockout system constructed in the laboratory. The phenotype of the mutant strain was studied. Compared with the wild type Brassicus, the growth rate of the mutant strain was slower, the cell morphology became larger, but the heterozygous strain had no obvious change. The sensitivity of the cell wall of heterozygous strain and variant strain to NaCl-SDS was not significantly changed, but the content of 尾 -glucan in the cell wall was significantly increased. This enriched the natural resources of 尾 -glucan. The results of animal experiments showed that the high content of 尾 -glucan mutant did not significantly improve the performance of experimental animals compared with wild-type strains. Its weight gain effect is not obvious; There was no obvious effect on the growth of immune organs of experimental animals, but the spleen index of experimental animals in variant strain group was the highest. The THP1 deletion mutant has the ability to resist Escherichia coli infection, and can reduce the mortality of experimental animals.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.7

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