本文選題：腸道微生物 + 菌群多樣性　； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：非洲獅(African Lions)是現(xiàn)存非洲最大的貓科動(dòng)物,屬食肉目(Carnivora),貓科(Felidae),豹屬(Panthera)。由于棲息地的破壞,現(xiàn)已出現(xiàn)小范圍的滅絕。而非洲獅疾病主要集中在口腔和消化道,因此開展不同季節(jié)非洲獅腸道菌群多樣性的研究對(duì)改變非洲獅腸道微生物組成、調(diào)節(jié)其優(yōu)勢(shì)菌群數(shù)量以及預(yù)防和治療腸道疾病十分必要。本研究首先采用PCR-DGGE技術(shù)結(jié)合熒光定量PCR技術(shù)研究不同季節(jié)非洲獅腸道菌群多樣性。經(jīng)PCR-DGGGE圖譜多樣性分析,發(fā)現(xiàn)冬季與春季組間腸道菌群相似性較高,夏季與秋季組間腸道菌群多樣性較高。對(duì)回收得到的13條差異條帶進(jìn)行測(cè)序分析,分別屬于Firmicutes菌門及Proteobacteria菌門,極少數(shù)為Bacteroidetes菌門。并將13條條帶中的5條特異性條帶設(shè)計(jì)其特異性引物進(jìn)行熒光定量PCR,結(jié)果顯示,這5種菌屬在不同季節(jié)非洲獅腸道中均存在,但數(shù)量差異顯著。為了對(duì)非洲獅腸道菌群多樣性進(jìn)行進(jìn)一步探索,本實(shí)驗(yàn)同時(shí)進(jìn)行宏基因組測(cè)序技術(shù),結(jié)果顯示,不同季節(jié)非洲獅腸道菌群中共有菌門為Firmicutes、Proteobacteria和Fusobacteria門。夏季非洲獅腸道內(nèi)含有大量的Bacteroidetes門,秋季和春季腸道內(nèi)含有少量的Chloroflexi門,進(jìn)一步對(duì)細(xì)菌屬水平進(jìn)行分析,春季優(yōu)勢(shì)菌屬為Collinsella和Lachnospiraceae,夏季則轉(zhuǎn)變?yōu)長(zhǎng)achnospiraceae和Campylobacter,而到了秋季Lachnospiracea逐漸被Campylobacter所取代,成為優(yōu)勢(shì)菌屬,冬季Escherichia-Shigella含量最多。最后為了進(jìn)一步探究差異菌株的功能活性,本實(shí)驗(yàn)利用了傳統(tǒng)培養(yǎng)的方法,篩選出具有功能活性的菌株,并對(duì)其進(jìn)行進(jìn)一步驗(yàn)證,Bacillus屬中Subtilis-partial和Bacillus-safensis均具有纖維素酶、淀粉酶和蛋白酶活性,而僅能通過(guò)宏基因組檢測(cè)到的Sporosarcina-globispora同時(shí)具有纖維素酶和淀粉酶活性,同為Sporosarcina屬的Sporosarcina-saromensis卻只有蛋白酶活性,Enterococcus屬中的Enterococcus-faecium和Enterococcus-mundtii均具有脂肪酶和木質(zhì)素酶活性。而既能在PCR-DGGE中檢測(cè)到又在宏基因組測(cè)序中被歸為優(yōu)勢(shì)菌屬的Bacillus-safensis既有纖維素酶活性又有蛋白酶活性。我們進(jìn)一步對(duì)它的產(chǎn)酶條件進(jìn)行優(yōu)化,優(yōu)化后Bacillus-safensis的發(fā)酵溫度為35℃,發(fā)酵時(shí)間為84h,pH為8,接種量為1%時(shí)產(chǎn)纖維素酶活性最高;發(fā)酵溫度為40℃,發(fā)酵時(shí)間為48h,pH為4,接種量為5%產(chǎn)蛋白酶活性最高。通過(guò)以上研究,可初步反應(yīng)不同季節(jié)非洲獅腸道菌群結(jié)構(gòu)及功能特征,這為非洲獅腸道菌群多樣性的研究及功能優(yōu)勢(shì)菌株的篩選奠定理論基礎(chǔ)。
[Abstract]:The African Lions are the largest cats in Africa, belonging to Carnivora, Felidaeae and Panthera. Due to habitat destruction, there is now a small scale of extinction. However, the diseases of African lions are mainly concentrated in the mouth and digestive tract. Therefore, the study on the diversity of intestinal flora of African lions in different seasons has changed the composition of the intestinal microbes of African lions. It is necessary to regulate the number of dominant bacteria and to prevent and treat intestinal diseases. In this study, PCR-DGGE technique and fluorescence quantitative PCR technique were used to study the diversity of intestinal flora of African lion in different seasons. It was found that the similarity of intestinal flora between winter and spring was higher than that between summer and autumn by analyzing the diversity of PCR-DGGGE map. The 13 differentially recovered bands were sequenced and analyzed. They belong to Firmicutes and Proteobacteria respectively, and a few belong to Bacteroidetes phylum. Five specific bands of 13 bands were designed for fluorescent quantitative PCR. The results showed that the five species of bacteria were found in the intestinal tract of African lions in different seasons, but there were significant differences in the number of them. In order to further explore the diversity of the intestinal flora of African lion, the macro genome sequencing technique was carried out at the same time. The results showed that the common phylum in the intestinal flora of African lion was Firmicutes-Proteobacteria and Fusobacteria phylum in different seasons. Bacteroidetes phylum was found in the intestines of African lions in summer and a small number of Chloroflexi phylum in their intestines in autumn and spring. The dominant bacteria were Collinsella and Lachnospiraceae in spring, Lachnospiraceae and Campylobacter in summer, and Lachnospiracea was gradually replaced by Campylobacter in autumn, and Escherichia-Shigella content was the highest in winter. Finally, in order to further explore the functional activity of different strains, we used the traditional culture method to screen the strains with functional activity, and further verified that Subtilis-partial and Bacillus-safensis have cellulase in the genus Bacillus. The activity of amylase and protease was found in Sporosarcina-globispora, which could only be detected by macrogenome, while in Sporosarcina-saromensis of Sporosarcina, only Enterococcus-faecium and Enterococcus-mundtii of Enterococcus had lipase and ligninase activity. Bacillus-safensis, which can be detected in PCR-DGGE and classified as dominant genus in macro genome sequencing, has both cellulase activity and protease activity. After optimization, the fermentation temperature of Bacillus-safensis was 35 鈩，

本文編號(hào)：1827994