五龄恩茅松毛虫幼虫的肠道好氧细菌多样性分析
Analysis of the diversity of intestinal aerobic bacteria from the 5th instar larvae of Dendrolimu kikuchii
王金华1，2**,李彪3,张武先1***,熊智1,赵丽芳1,王海林1
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作者单位：1 西南林业大学西南山地森林资源保育与利用省部共建教育部重点实验室昆明650224；2 云南农业大学植物保护学院昆明650201； 3 云南瑞升烟草技术（集团）有限公司 昆明650106
中文关键词：思茅松毛虫, 肠道细菌, 多样性, 鉴定
英文关键词：Dendrolimus kikuchii, gut microbiology, diversity, identification
中文摘要：采用16s rDNA通用引物进行PCR扩增，16s rDNA扩增片段经寡位点酶组合（Hae Ⅲ + Hind Ⅲ）和多位点酶组合（HinfⅠ+ TaqⅠ）酶切后得到
ARDRA指纹图谱，ARDRA指纹图谱通过MVSP软件进行聚类分析（UPGMA），并对5大类群的代表菌株进行测序，研究了5龄思茅松毛虫Dendrolimu
kikuchii Matsumura肠道细菌的多样性。结果表明：寡位点酶组合（Hae Ⅲ + Hind Ⅲ）或多位点酶组合（HinfⅠ+ TaqⅠ）酶切得到的ARDRA
指纹图谱不能反映5龄思茅松毛虫肠道细菌的多样性，两组酶切结果组合后可以充分反映5龄思茅松毛虫肠道细菌的多样性。通过测序可知，5
龄思茅松毛虫的肠道内主要定植产酸克雷伯氏杆菌（Klebsiella oxytoca）、雷金斯堡约克氏菌(Yokenella regensburgei)、克雷伯氏杆菌（
Klebsiella peneumoniae）、弗氏柠檬酸杆菌（Citrobacter freundii）、苏云金芽孢杆菌（Bacillus thuringiensis），多样性丰富，它们
形成的肠道微生物生态系统可以抵制外来菌对恩茅松毛虫的侵害，维持恩茅松毛虫的健康。
英文摘要：In order to investigate the diversity of the intestinal aerobic bacteria of the 5th instar larvae of Dendrolimus kikuchii
Matsumura the characteristic ARDRA fingerprint of the larvae’s intestinal bacteria was obtained by amplifying 16S rDNA with
a general primer and digesting the 16S rDNA PCR amplicon (about 15 kb) with four restriction enzymes (Hae Ⅲ, Hind Ⅲ,
HinfⅠ and TaqⅠ). ARDRA patterns were analyzed by MVSP software using the UPGMA method，and the full complement of 5
representative strains’ 16S rDNA were sequenced and blasted in GenBank. Although a few enzyme combinations (Hae Ⅲ + Hind
Ⅲ and Hinf Ⅰ + Taq Ⅰ) could not completely digest the 16S rDNA PCR amplicon, the diversity of intestinal bacteria could
be inferred from the results obtained from all four restriction enzymes. The results of phylogenetic analysis based on
sequencing show that intestinal aerobic bacteria from the 5th instar larvae of D. kikuchii include Klebsiella oxytoca,
Yokenella regensburgei, Klebsiella peneumoniae, Citrobacter freundii and Bacillus thuringiensis. This diverse intestinal
microbial ecosystem may help protect larvae from invasion by invasive pathogenic bacteria.