长寿老人肠道双歧杆菌菌群群落结构及其分离株多位点序列分型的研究

Although Bifidobacterium as commercial starter bacteria is widely used in food processing, the research on the genetic diversity and phylogenetic relationships among Bifidobacterium isolates is relatively backward. A total of 40 long-lived people (age >90 y) living in nursing home located in Wuhan and Long Life Village in Enshi are recruited, and fresh fecal samples are collected. A combination of polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) is used to examine the structure of Bifidobacterium in the fecal of the subjects, and then the species of Bifidobacterium are enumerated by Real-time quantitative PCR method. Meanwhile, Bifidobacterium isolates are isolated using pure culture method from the fecal of long-lived people, after that those isolates are identified by morphology and molecular biology. To determine the genetic diversity and phylogenetic relationships among Bifidobacterium isolates, strains isolated from the fecal of long-lived people living in different geography are subjected to multilocus sequence typing (MLST). The sequences of 8~12 housekeeping genes are acquired by Bidirectional sequencing technology, forward and reverse sequences are trimmed, aligned, and analyzed by molecular biology software, an allele number is given to each distinct combination of alleles of these genes, and each allele number is assigned a sequence type (ST). By comparing the difference of alleles sequences among strains, systematic realize the evolutionary relationship of Bifidobacterium isolates from different geography, and provide the basis of genetic underpinnings for the following research and commercial development of Bifidobacterium strains.

双歧杆菌是食品加工业中常用的发酵剂菌种之一，然而目前我国在其遗传进化关系研究方面开展的工作相对滞后。本项目拟从武汉市市区养老院和恩施市长寿村各采集长寿老人（≥90岁）粪便样品20份，首先，使用双歧杆菌属特异性引物对粪便微生物进行扩增，在进行 PCR-DGGE 分析明确双歧杆菌菌群构成的基础上，根据特定菌群的基因序列设计适合的引物进行 RT-PCR 扩增，得到长寿老人肠道中双歧杆菌菌群的数量信息。同时，采用稀释涂布平板法对双歧杆菌菌株进行分离、纯化，并采用分子生物学方法对已分离的菌株进行分类鉴定。最后，选取上述菌株进行多位点序列分型研究，通过双向测序技术获得 8~12 个持家基因的序列，将其拼接、核对后用分子生物学软件构建等位基因图谱并确立序列型，进而对不同地域长寿老人源双歧杆菌菌种进行分型，系统分析菌株间的系统发育进化关系，从而为双歧杆菌后续的研究、商业化开发和利用奠定坚实的遗传学基础。

本课题对生活在襄阳和恩施地区长寿老人的肠道微生物群落结构进行了研究，同时对双歧杆菌进行了分离鉴定并解析了分离株的遗传进化关系。.（1）本部分研究选取22例在恩施地区和20例在襄阳地区生活长寿老人的粪便样品作为研究对象，以 Miseq高通量测序技术结合多变量统计学方法检测细菌 16S rRNA基因V1-V3 区的多样性。虽然基于UniFrac的PcoA分析和UPGMA分析均显示两个人群的样本存在交叠现象，但MANOVA分析显示其肠道菌群群落结构差异显著（P < 0.05）。襄阳地区长寿老人肠道菌群的多样性、丰度和组内平均UniFrac 距离均大于恩施地区（P < 0.05）。进一步研究发现，Alistipes、Collinsella、Coprococcus、Eubacterium、Roseburia、Faecalibacterium、Gemmiger和Prevotella在襄阳地区长寿老人肠道菌群中的含量明显偏高（P < 0.05）。.（2）DGGE结果表明：长寿老人肠道中的双歧杆菌主要为B. dentium、B. longum和B. pseudocatenulatum。RT-PCR结果表明：襄阳地区长寿老人肠道中双歧杆菌的含量显著高于恩施地区。分离出的91 株双歧杆菌菌株，分别为B. dentium 6株；B. breve1株； B. faecale 5株；B. pseudocatenulatum 22株；B. bifidum 8株； B. adolescentis 5株；B. longum 44株。.（3）本研究进一步以分离自长寿老人肠道的40株长双歧杆菌为研究对象，利用看家基因clpC、fusA、ileS、purF和rplB构建MLST方案，通过对试验菌株的基因多样性进行研究进而推演菌株间的亲缘关系和进化历程，结果发现不同地区长寿老人来源的菌株不存在ST分型。.通过本课题的实施在为商业化双歧杆菌的开发利用提供菌株支持的同时，亦可为其后续的研究、开发和利用奠定坚实的遗传学基础。