贡嘎蝠蛾幼虫肠道细菌多样性分析

Analysis of the bacterial diversity in intestines of Hepialus gonggaensis larae

摘要:

【目的】对实验室养殖条件下的重要经济昆虫冬虫夏草寄主—贡嘎蝠蛾（Hepialus gonggaensis, Hg）幼虫肠道微生物群落的多样性进行了研究。【方法】采用常规分离培养与分子鉴定的方法和基于16S rRNA作为分子标记的变性梯度凝胶电泳（denaturing gradient gel electrophoresis, DGGE）的方法。【结果】用常规分离与分子鉴定方法获得8个属的细菌类群，其中肠杆菌属（Enterobacter）是优势菌群，肉食杆菌属（Carnobacterium）是次优势菌群。对通过DGGE方法得到的11条16S rRNA优势条带序列进行了比对和系统进化树分析，结果表明肉食杆菌属（Carnobacterium）的丰度最高，是肠道细菌中主要的优势菌群，芽孢杆菌属（Bacillus）是次优势菌群。DGGE图谱还显示Hg幼虫不同虫龄肠道细菌菌群的结构存在差异，推测可能与其发育生理状态的差异有关系。【结论】结合常规分离法与DGGE法能够更有效的分析肠道微生物的多样性，获得更多更全面的微生物多样性信息。

关键词:贡嘎蝠蛾幼虫;16S rRNA;常规分离培养;变性梯度凝胶电泳;群落多样性

Abstract:

[Objective] We investigated the intestinal microbial diversity in the larval gut of Hepialus gonggaensis, an economically important insect. [Methods] We used morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis method, and the molecular method of PCR-DGGE (denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V3 region gene. [Results] By the traditional isolation method, 8 genera of bacteria were identified from 11 isolated bacterial populations. The dominant bacteria in intestine belonged to enterobacter. By 16S rRNA V3 region gene DGGE method, eleven distinct bands were obtained from 16S rDNA amplificons. The bands were purified, sequenced. The sequences aligned with GenBank database and showed that they were belonged to 8 different genera of bacteria. Phylogenetic analysis showed that the sequences of bacteria belonged to the Proteobacteria and Firmicutes. The most dominant bacteria group was Carnobacterium in the gut and Bacillus followed by it. The different patterns were observed in different instars larvae guts from DGGE profiles, which might be related to their physiological development stages. [Conclusion] 8 genera were obtained from intestine of H. gonggaensis by traditional culturing method and 16S rDNA analysis method respectively, but the two groups were not exactly same, and the dominant group was different also. This suggested that a combination of molecular and traditional culturing methods can be used to analyze and monitor the diversity of intestinal microflora effectively, and that will give us more information of microorganism diversity.

Key words:Hepialus gonggaensis;16S rRNA; normal isolation culture; denaturing gradient gel electrophoresis (DGGE); specie diversity