本文關(guān)鍵詞： 布拉氏酵母菌 β-葡聚糖 基因敲除 益生功能 基因工程　出處：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：布拉氏酵母菌(S.boulardii)是法國(guó)科學(xué)家Henri Boulard于1920年從印尼荔枝等水果中分離得到的一種益生酵母菌。目前,布拉菌作為微生態(tài)制劑在歐美等國(guó)家廣泛用于防治旅行者和兒童的各種腹瀉。畜牧業(yè)上,在飼料中添加布拉氏酵母菌制劑以提高動(dòng)物的生產(chǎn)性能來(lái)提升經(jīng)濟(jì)效益已得到包括中國(guó)、歐盟在內(nèi)的世界上許多國(guó)家的認(rèn)可。隨著人們對(duì)健康和生態(tài)環(huán)境的高度重視及在畜禽養(yǎng)殖中的進(jìn)一步研究,布拉菌在畜牧業(yè)和醫(yī)學(xué)上必將發(fā)揮更大的作用,同時(shí)也會(huì)有更廣闊的應(yīng)用前景。布拉氏酵母菌作為最具有發(fā)展?jié)摿Φ囊嫔?其細(xì)胞壁的主要組成成分β-葡聚糖在布拉菌作為益生菌整體的作用中起著關(guān)鍵作用。酵母菌β-葡聚糖是一種具有增強(qiáng)免疫力活性的多糖,占布拉氏酵母菌細(xì)胞壁組成的50%以上,因此,布拉菌無(wú)疑是β-葡聚糖的重要來(lái)源之一。然而,對(duì)于β-葡聚糖功能的研究,大都是針對(duì)分離純化的β-葡聚糖進(jìn)行的。β-葡聚糖作為布拉菌細(xì)胞壁的主要組成成分,在布拉菌作為益生菌整體的作用中,不同β-葡聚糖含量與布拉菌的益生功能的影響卻未見(jiàn)研究和報(bào)道。因此,本研究主要是通過(guò)Cre-loxP基因敲除系統(tǒng)對(duì)目標(biāo)基因進(jìn)行基因敲除,構(gòu)建一個(gè)布拉菌細(xì)胞壁高含量β-葡聚糖的變異菌株,可豐富β-葡聚糖的自然資源,為工業(yè)上大量提取β-葡聚糖提供了原材料。同時(shí),研究敲除目標(biāo)基因后對(duì)布拉菌表型的影響,并通過(guò)體外及動(dòng)物實(shí)驗(yàn)研究β-葡聚糖在布拉菌作為益生菌的益生功能中的作用,以及布拉菌細(xì)胞壁β-葡聚糖含量的變化對(duì)其益生功能、非特異性免疫能力、抗感染能力及生產(chǎn)性能的影響,探討布拉菌在分子水平上的作用機(jī)理,為進(jìn)一步改善其益生特性打下基礎(chǔ)和提供技術(shù)支持。本研究根據(jù)研究的目的和意義選出1個(gè)可明顯影響布拉氏酵母菌細(xì)胞壁β-葡聚糖含量的基因THP1,通過(guò)實(shí)驗(yàn)室構(gòu)建的適合布拉菌的Cre-loxP基因敲除系統(tǒng)成功篩選出布拉菌細(xì)胞壁高含量β-葡聚糖的變異菌株Sb-thp1。對(duì)雜合子菌株、變異株的表型進(jìn)行研究發(fā)現(xiàn),和野生型布拉菌相比,變異株的生長(zhǎng)速度減慢,細(xì)胞形態(tài)變圓變大,而雜合子菌株無(wú)明顯變化;雜合子菌株、變異株細(xì)胞壁對(duì)剛果紅、NaCl、SDS的敏感性無(wú)明顯變化,但細(xì)胞壁β-葡聚糖的含量明顯升高,這豐富了β-葡聚糖的自然資源。動(dòng)物實(shí)驗(yàn)結(jié)果表明,布拉菌細(xì)胞壁高含量β-葡聚糖變異株和野生型布拉菌相比沒(méi)有顯著提高試驗(yàn)動(dòng)物的生產(chǎn)性能,其增重效果不明顯;對(duì)試驗(yàn)動(dòng)物免疫器官的生長(zhǎng)沒(méi)有明顯的促進(jìn)作用,但變異株組的試驗(yàn)動(dòng)物的脾臟指數(shù)最高;布拉菌THP1缺失變異株有一定的抗大腸桿菌感染的能力,能降低試驗(yàn)動(dòng)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S816.7

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本文編號(hào)：1450956