动物营养学报 >

2018 , Vol. 30 >Issue 2: 649 - 657

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.02.030

分子营养 Molecular Nutrition

聚合酶链式反应-变性梯度凝胶电泳技术分析1只死亡野生川金丝猴胃肠道菌群多样性

展开
  • 1. 四川农业大学动物医学院动物微生态研究中心, 成都 611130;
    2. 成都动物园, 成都 610081;
    3. 四川省畜牧科学研究院, 成都 610066
曾燕(1987-),女,四川内江人,博士研究生,从事动物微生态研究。E-mail:zengyan88@stu.sicau.edu.cn

收稿日期: 2017-07-14

  网络出版日期: 2018-02-02

基金资助

成都大熊猫繁育研究基金会项目（CPF2015-06）；国家自然科学基金（31672318）

收起

Bacteria Diversity in Gastrointestinal Tract of a Dead Wild Rhinopithecus roxellanae Analyzed by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis Technology

Expand
  • 1. Institute of Animal Microecology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
    2. Chengdu Zoo, Chengdu 610081, China;
    3. Sichuan Animal Science Institute, Chengdu 610066, China

Received date: 2017-07-14

  Online published: 2018-02-02

Fold

摘要

本试验旨在对1只死亡野生川金丝猴的胃肠道菌群多样性及其克隆测序条带的系统进化树进行分析。取死亡野生川金丝猴胃肠道内容物，进行聚合酶链式反应（PCR）-变性梯度凝胶电泳（DGGE）分析，结合条带的克隆测序、聚类分析和主成分分析（PCA）检测菌群多样性并构建系统进化树。结果显示：1）野生川金丝猴整个胃肠道栖息着大量细菌，且来自胃、小肠的样品聚为一大簇，来自大肠的样品聚为一簇，而来自粪便的样品单独聚为一簇。2）从DGGE图谱上共回收18个条带，细菌种类鉴定主要是5个菌门，分别为变形菌门（Proteobacteria，38.89%）、厚壁菌门（Firmicutes，22.22%）、拟杆菌门（Bacteroidetes，5.56%）、放线菌门（Actinobacteria，5.56%）、疣微菌门（Verrucomicrobia，5.56%）和不可培养菌（uncultured bacterium，22.22%）。其中，变形菌门和厚壁菌门分布于整个胃肠道。3）菌群的系统进化树分析表明，仅有1种不可培养菌与已鉴定的粪肠球菌进化分类相似，而其他不可培养菌与已知的菌种进化分支差异较大，说明在野生川金丝猴的胃肠道中仍有大量的菌群信息未被鉴定。结果提示，本试验测定的1只死亡野生川金丝猴的胃肠道优势菌群为变形菌门，菌群多样性随着胃肠道由前至后的顺序呈现高—低—高的趋势。

关键词： 川金丝猴; 胃肠道菌群; PCR-DGGE; 克隆测序; 系统进化树

本文引用格式

曾燕, 牛李丽, 邓家波, 王强, 余建秋, 李杨, 周梦佳, 徐帅, 林奕岑, 曾东, 倪学勤 . 聚合酶链式反应-变性梯度凝胶电泳技术分析1只死亡野生川金丝猴胃肠道菌群多样性[J]. 动物营养学报, 2018 , 30(2) : 649 -657 . DOI: 10.3969/j.issn.1006-267x.2018.02.030

Abstract

The aim of this experiment was to evaluate the bacteria diversity in gastrointestinal tract (GIT) of a dead wild Rhinopithecus roxellanae, and to analyze the phylogenetic tree of cloning sequencing bands. The GIT contents of a dead wild Rhinopithecus roxellanae were collected, and the polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technology with the cloning sequencing, cluster analysis and principal component analysis (PCA) of bands were used to detect the bacterial diversity and to build the phylogenetic tree. The results showed as follows:1) numerous bacteria were obtained from the GIT of wild Rhinopithecus roxellanae. Samples from the stomach, duodenum, jejunum and ileum clustering together, samples from the cecum, colon and rectum clustering together, and samples from the faeces single clustering together. 2) The 18 identified DGGE bands were belong to five phylum, they were Proteobacteria (38.89%), Firmicutes (22.22%), Bacteroidetes (5.56%), Actinobacteria (5.56%), Verrucomicrobia (5.56%) and uncultured bacterium (22.22%). The Proteobacteria and Firmicutes were detected along the GIT. 3) The phylogenetic tree analysis showed that only one of uncultured bacterium was similar to the identified classification of Enterococcus faecalis, while the other three uncultured bacteria had a significant difference with the known branch of the bacteria. This indicated that a large number of flora information was unkowned in the GIT of the wild Rhinopithecus roxellanae. The results suggest that Proteobacteria is predominant in the GIT of a dead wild Rhinopithecus roxellanae, and the bacteria diversity shows a tendency of high-low-high according to the GIT from front to back.

Key words： Rhinopithecus roxellanae; gastrointestinal tract bacteria; PCR-DGGE; cloning sequencing; phylogenetic tree

参考文献

[1] 彭燕章,叶智彰,张耀平,等.金丝猴分类及系统发育关系[J].动物学研究,1988,9(3):239-248.

[2] 全国强,谢家骅.金丝猴研究[M].上海:上海科技教育出版社,2002.

[3] ZHOU X M,WANG B S,PAN Q,et al.Whole-genome sequencing of the snub-nosed monkey provides insights into folivory and evolutionary history[J].Nature Genetics,2014,46(12):1303-1310.  

[4] 金崑,刘世荣,顾志宏,等.我国川金丝猴的重要栖息地及自然保护区[C]//中国科协2005年学术年会.乌鲁木齐:中国林学会,2005.

[5] STUMPF R M,GOMEZ A,AMATO K R,et al.Microbiomes,metagenomics,and primate conservation:new strategies,tools,and applications[J].Biological Conservation,2016,199:56-66.

[6] PENNISI E.How do gut microbes help herbivores?Counting the ways[J].Science,2017,355(6322):236.

[7] GEVAZA-TORSKY N,SEFIK E,KUA L,et al.Mining the human gut microbiota for immunomodulatory organisms[J].Cell,2017,168(5):928-943.  

[8] 刘燕,郑常明,李林海,等.一只半野生病死川金丝猴肠道微生物多样性分析[J].野生动物学报,2017,38(2):194-199.

[9] 杨曼,兰阿峰,郭素芬,等.免培养法研究野生川金丝猴肠道内生细菌多样性[J].微生物学通报,2014,41(8):1605-1612.

[10] MUYZER G,DE WAAL E C,UITTERLINDEN A G.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J].Applied and Environmental Microbiology,1993,59(3):695-700.

[11] VIGNIERI S.Vanishing fauna[J].Science,2014,345(6195):392-395.  

[12] YOUNG H S,DIRZO R,HELGEN K M,et al.Declines in large wildlife increase landscape-level prevalence of rodent-borne disease in Africa[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(19):7036-7041.  

[13] EVANS R,ALESSI A M,BIRD S,et al.Defining the functional traits that drive bacterial decomposer community productivity[J].ISME Journal,2017,11(7):1680-1687.  

[14] BRADLEY P H,POLLARD K S.Proteobacteria explain significant functional variability in the human gut microbiome[J].Microbiome,2017,5(1):36.

[15] LUEDDE M,WINKLER T,HEINSEN F A,et al.Heart failure is associated with depletion of core intestinal microbiota[J].ESC Heart Failure,2017,4(3):282-290.  

[16] MCKNNEY E A,RODRIGO A,YODER A D.Patterns of gut bacterial colonization in three primate species[J].PLoS One,2015,10(5):e0124618.

[17] 王剑,王强,曾东,等.健康和腹泻川金丝猴粪样菌群比较分析[J].中国兽医学报,2015,35(8):1232-1238.

[18] AIVELO T,LAAKKONEN J,JERNVALL J,et al.Population-and individual-level dynamics of the intestinal microbiota of a small primate[J].Applied and Environmental Microbiology,2016,82(12):3537-3545.  

[19] SCHUSTER S C.Next-generation sequencing transforms today's biology[J].Nature Methods,2008,5(1):16-18.  

[20] KOHL K D,SKOPEC M M,DEARING M D.Captivity results in disparate loss of gut microbial diversity in closely related hosts[J].Conservation Physiology,2014,2(1):cou009.

[21] YILDIRIM S,YEOMAN C J,SIPOS M,et al.Characterization of the fecal microbiome from non-human wild primates reveals species specific microbial communities[J].PLoS One,2010,5(11):e13963.

[22] SUN B H,WANG X,BERNSTEIN S,et al.Marked variation between winter and spring gut microbiota in free-ranging Tibetan Macaques (Macaca thibetana)[J].Science Reports,2016,6:26035.

[23] HALE V L,TAN C L,NIU K F,et al.Diet versus phylogeny:a comparison of gut microbiota in captive Colobine monkey species[J].Microbial Ecology,2017:1-13.

[24] TROPINI C,EARLE K A,HUANG K C,et al.The gut microbiome:connecting spatial organization to function[J].Cell Host & Microbe,2017,21(4):433-442.  

[25] 陈嘉绩,陆桐,刘建遂,等.川金丝猴胃的观察[J].兽类学报,1995,15(3):176-180.

[26] GU S H,CHEN D D,ZHANG J N,et al.Bacterial community mapping of the mouse gastrointestinal tract[J].PLoS One,2013,8(10):e74957.

[27] SEEDORF H,GRIFFIN N W,RIDAURA V K,et al.Bacteria from diverse habitats colonize and compete in the mouse gut[J].Cell,2014,159(2):253-266.  

[28] KATSOWICH N,ELBAZ N,PAL R R,et al.Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria[J].Science,2017,355(6326):735-739.  

[29] 吕颜枝,康永刚,朱广琴,等.猕猴群暴发细菌性痢疾的诊治[J].畜牧与兽医,2012,44(9):53-54.

[30] BAKSHI U,SARKAR M,PAUL S,et al.Assessment of virulence potential of uncharacterized Enterococcus faecalis strains using pan genomic approach-identification of pathogen-specific and habitat-specific genes[J].Scientific Reports,2016,6:38648.

[31] 简平,王强,王剑,等.不同年龄段川金丝猴肠道菌群结构差异分析[J].动物营养学报,2015,27(4):1302-1309.

[32] HUG L A,BAKER B J,ANANTHARAMAN K,et al.A new view of the tree of life[J].Nature Microbiology,2016,1:16048.

[33] 赵晗旭.不同野生动物肠道微生物多样性分析及功能初步研究[D].硕士学位论文.长春:吉林农业大学,2015.
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2