不同季节圈养非洲狮肠道微生物多样性分析及功能学初步研究

发布时间：2018-05-01 05:38

  本文选题：肠道微生物 + 菌群多样性　； 参考：《吉林农业大学》2017年硕士论文

【摘要】：非洲狮(African Lions)是现存非洲最大的猫科动物,属食肉目(Carnivora),猫科(Felidae),豹属(Panthera)。由于栖息地的破坏,现已出现小范围的灭绝。而非洲狮疾病主要集中在口腔和消化道,因此开展不同季节非洲狮肠道菌群多样性的研究对改变非洲狮肠道微生物组成、调节其优势菌群数量以及预防和治疗肠道疾病十分必要。本研究首先采用PCR-DGGE技术结合荧光定量PCR技术研究不同季节非洲狮肠道菌群多样性。经PCR-DGGGE图谱多样性分析,发现冬季与春季组间肠道菌群相似性较高,夏季与秋季组间肠道菌群多样性较高。对回收得到的13条差异条带进行测序分析,分别属于Firmicutes菌门及Proteobacteria菌门,极少数为Bacteroidetes菌门。并将13条条带中的5条特异性条带设计其特异性引物进行荧光定量PCR,结果显示,这5种菌属在不同季节非洲狮肠道中均存在,但数量差异显著。为了对非洲狮肠道菌群多样性进行进一步探索,本实验同时进行宏基因组测序技术,结果显示,不同季节非洲狮肠道菌群中共有菌门为Firmicutes、Proteobacteria和Fusobacteria门。夏季非洲狮肠道内含有大量的Bacteroidetes门,秋季和春季肠道内含有少量的Chloroflexi门,进一步对细菌属水平进行分析,春季优势菌属为Collinsella和Lachnospiraceae,夏季则转变为Lachnospiraceae和Campylobacter,而到了秋季Lachnospiracea逐渐被Campylobacter所取代,成为优势菌属,冬季Escherichia-Shigella含量最多。最后为了进一步探究差异菌株的功能活性,本实验利用了传统培养的方法,筛选出具有功能活性的菌株,并对其进行进一步验证,Bacillus属中Subtilis-partial和Bacillus-safensis均具有纤维素酶、淀粉酶和蛋白酶活性,而仅能通过宏基因组检测到的Sporosarcina-globispora同时具有纤维素酶和淀粉酶活性,同为Sporosarcina属的Sporosarcina-saromensis却只有蛋白酶活性,Enterococcus属中的Enterococcus-faecium和Enterococcus-mundtii均具有脂肪酶和木质素酶活性。而既能在PCR-DGGE中检测到又在宏基因组测序中被归为优势菌属的Bacillus-safensis既有纤维素酶活性又有蛋白酶活性。我们进一步对它的产酶条件进行优化,优化后Bacillus-safensis的发酵温度为35℃,发酵时间为84h,pH为8,接种量为1%时产纤维素酶活性最高;发酵温度为40℃,发酵时间为48h,pH为4,接种量为5%产蛋白酶活性最高。通过以上研究,可初步反应不同季节非洲狮肠道菌群结构及功能特征,这为非洲狮肠道菌群多样性的研究及功能优势菌株的筛选奠定理论基础。
[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，

本文编号：1827994