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

作　　者：王金华[1,2] 李彪 张武先[1] 熊智[1] 赵丽芳[1] 王海林[1] 

机构地区：[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

出　　处：《应用昆虫学报》2013年第1期230-234,共5页Chinese Journal of Applied Entomology

基　　金：云南省森林灾害预警与控制重点实验室开放基金(ZK09A102);国家自然科学基金(31100007);西南林业大学大型仪器设备共享平台资助

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(HaeⅢ+HindⅢ)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(HaeⅢ+HindⅢ)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。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 1.5 kb） with four restriction enzymes （Hae III , Hind 11I , Hinf I and Taq I ）. 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 llI ＋ Hind m and Hinf ｜ ＋ 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.

关 键 词：思茅松毛虫 肠道细菌 多样性 鉴定 

分 类 号：S763.421[农业科学—森林保护学]