共生菌群在熊蜂生长发育过程中的动态变化

doi:10.3864/j.issn.0578-1752.2014.10.017

摘要/Abstract

摘要： 【目的】了解人工饲养的兰州熊蜂（Bombus lantschouensis）肠道内共生菌群的组成，探明主要肠道共生菌群在熊蜂生长发育过程中的变化规律，为进一步开展熊蜂共生菌功能的研究奠定基础。【方法】以兰州熊蜂肠道总DNA为模板，使用细菌通用的774F和1391R引物进行PCR扩增，构建细菌16S rDNA文库，挑取单克隆菌落测序，测得序列去除chimera后，以序列相似性97%为标准，划分分类操作单元（operational taxonomic units，OTU），采用BLASTn进行序列同源性分析，确定细菌种类，分析熊蜂肠道菌群的组成。根据克隆文库测得的Gilliamella apicola和Snodgrassella alvi细菌16S rDNA序列设计特异性引物。用G. apicola和S. alvi的特异性引物进行PCR扩增，构建重组子质粒，构建好的质粒经浓度测定后，10倍梯度稀释成5个浓度梯度，进行荧光定量PCR检测，绘制标准曲线。以兰州熊蜂的卵、幼虫、蛹以及0、5、10、15、20日龄的工蜂肠道DNA为模板，采用熊蜂β-actin为内参基因，对样品中的共生菌G. apicola和S. alvi进行荧光定量PCR分析，并比较不同日龄、不同虫态每微升肠道基因组DNA样品中检测到的细菌16S rDNA基因的拷贝数，分析共生菌数量在熊蜂生长发育过程中的变化。不同日龄、不同虫态之间相对表达量的显著性差异用软件SPSS19.0的单因素ANOVA方差分析进行统计。【结果】从文库中随机挑选213个单克隆进行测序，经过Chimeras分析后，共得到202个有效序列，这些序列共划分为16个OTU。测得的序列与登录的相应细菌16S rDNA序列的相似性在93%—99%。在克隆文库测得的细菌16S rDNA序列中，G. apicola占45%、S. alvi占30%、Bifidobacterium占10%、Fructobacillus fructose占5%、Lactobacillus占2%、Flavobacterium aciduliphilum占2%，其他细菌占6%。其中G. apicola和S. alvi为兰州熊蜂肠道内的主要共生菌，qPCR结果表明两种共生菌在不同日龄、不同虫态的熊蜂肠道内都能检测到，两种细菌的数量在熊蜂发育过程中的变化趋势相似，即先增加后减少，最后达到稳定状态。G. apicola和S. alvi在熊蜂的卵、幼虫和蛹中数量都较少，在5日龄时的数量达到峰值，显著高于其他日龄，之后又逐渐减少，在15日龄后趋于稳定，第15、20日龄之间差异不显著。【结论】人工饲养的兰州熊蜂肠道中主要有4个种属的常见共生菌：G. apicola、S. alvi、F. fructosus和Bifidobacterium，其中G. apicola和S. alvi是其体内的优势菌。G. apicola和S. alvi在熊蜂中都具有水平和垂直传播的特性，两种共生菌在熊蜂的卵、幼虫和蛹中都检测到，但数量较少，工蜂出房后细菌大量增殖并在出房15 d左右形成稳定的共生菌群。熊蜂生长发育过程中体内共生菌数量的变化可能与这两种细菌对熊蜂的作用有关。

关键词: 熊蜂 , 共生菌 , 肠道微生物 , 荧光定量PCR

Abstract: 【Objective】 The objectives of this study are to examine microbial communities from the digestive tract of Bombus lantschouensis reared in the laboratory, and to analyze the specific bacteria spatio-temporally in different developmental stages of B. lantschouensis, which is the important foundation and basis for further studying the function of symbiotic bacteria in bumblebee. 【Method】The fragment of the bacterial 16S rDNA gene was PCR-amplified with the universal eubacterial primers 774F and 1391R and a 16S rDNA clone library of gut bacteria was constructed. Each single clone was picked and sequenced. The sequences were checked for chimeras, sequences of chimeric origin were removed from further analysis. Sequences obtained were analyzed by BLASTn and matched with valid reference sequences in the NCBI (National Center for Biotechnology Information) to determine the bacterial species types. The 16S rDNA gene primers of the specific bacteria Gilliamella apicola and Snodgrassella alvi were designed based on sequencing of bacterial taxonomic groups obtained from the clone library, then constructed recombinant plasmid using the specific bacteria primers and diluted the plasmid to 10 folds series for 5 concentration gradients after concentration determination. Total genomic DNA was extracted from egg, larva, pupa, and 0, 5th, 10th, 15th, 20th days worker bees. The bumblebee β-actin gene was selected as the reference gene, the quantitative real-time PCR (qPCR) was used to explore the quantity variation of symbionts during the different developmental stages of B. lantschouensis.【Result】A total of 213 clones were picked and sequenced, after Chimeras checking, 202 sequences were obtained and then divided into 16 OTUs. The sequence similarity with GenBank sequences was between 93%-99%. The phylogenetic analysis found that the sequences fell into 6 major lineages within the domain bacteria. Among them, 45% and 30% were respectively G. apicola and S. alvi, 10% was Bifidobacterium, 5% was Fructobacillus fructose, 2% was Lactobacillus, 2% was Flavobacterium aciduliphilum, and 6% was mixture of uncommon bacteria types. The qPCR result showed that G. apicola and S. alvi were detected in all development stages, the two bacterial species were similar in the quantity variation patterns with decreasing after increasing, finally reached the steady state. The quantity of G. apicola and S. alvi in egg, larva and pupa was very low, in 5th day significantly higher than those in other development stages, and then gradually reduced, stabilized after 15th day, the bacteria quantity in 20th day showed no difference with 15th day.【Conclusion】Four common symbiotic bacteria including G. apicola, S. alvi, F. fructose and Bifidobacterium were identified in the digestive tract of B. lantschouensis reared in laboratory. G. apicola and S. alvi are dominant bacterium among them. G. apicola and S. alvi both have horizontal and vertical transmission. The quantity of G. apicola and S. alvi in egg, larva and pupa was very low, and increased rapidly after worker bees emergence, stabilized after 15 days. The temporal variation of two dominant bacteria in B. lantschouensis may be related to the function of symbiotic bacteria in bumblebee.

Key words: bumblebee , symbiotic bacteria , gut microbiota , qPCR

徐龙龙, 吴杰, 郭军, 李继莲. 共生菌群在熊蜂生长发育过程中的动态变化[J]. 中国农业科学, 2014, 47(10): 2030-2037.

XU Long-Long, WU Jie, GUO Jun, LI Ji-Lian. Dynamic Variation of Symbionts in Bumblebees During Hosts Growth and Development[J]. Scientia Agricultura Sinica, 2014, 47(10): 2030-2037.

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