思茅松毛虫四龄幼虫肠道好氧细菌的ARDRA分析及鉴定  

Amplified ribosomal DNA restriction analysis and identification of intestinal aerobic bacteria from 4th instar larva of Dendrolimu kikuchii

作　　者：孙佑赫[1] 熊智[1] 王金华[1] 张武先[1] 王海林[1] 

机构地区：[1]西南林业大学,昆明650224

出　　处：《应用昆虫学报》2012年第6期1618-1622,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)

摘　　要：从思茅松毛虫Dendrolimu kikuchii Matsumura4龄幼虫肠道环境中分离、纯化、培养,获得11株好氧细菌菌株。以细菌基因组DNA为模板,用细菌通用引物(27f、1492r)对模板进行PCR扩增,并用4种限制性内切酶HaeⅢ、HindⅢ、HinfⅠ、TaqⅠ对PCR产物进行ARDRA(amplified rDNA restriction analysis)多态性分析,在84%的相似水平上可分成6大类群,多样性丰富。对6大类群的代表菌株进行16SrDNA序列测定,分析表明:分离到的11株好氧细菌中4N05、4N08和4N11归属克雷伯氏杆菌属(Klebsiellasp.),4N02归属Lysinibacillus属;4N03和4N09确定到种,分别是γ-变形杆菌(Gamma proteobacterium)和枯草芽孢杆菌(Bacillus subtilis strain);4N04、4N06、4N07和4N01、4N10也可以确定到种,其中前3株是短短芽孢杆菌(Brevibacillus brevisstrain),后2株是沼泽短芽孢杆菌(Brevibacillus limnophilusstrain),并且这5株菌都归属短芽孢杆菌属(Brevibacillus sp.)。通过研究,既可以为松毛虫的生物防治提供微生态理论依据,又丰富了微生物资源库。11 strains of aerobic bacteria were isolated from the intestine of Dendrolimu kikuchii Matsumura using the plating method. Bacterial genomic DNA was used as templates. A new method of analyzing the genetic diversity of intestinal aerobic bacteria was established using the PCR technique （primer27f, 1492r） and ARDRA patterns, which used enzyme digestion （HaeⅢ and HindⅢ , Hinf I and Taq I ） of cloned 16S rDNA gene sequences. The results show that 11 strains could be divided into 6 groups at a similarity level of 84%. The results prove that Dendrolimu kikuchii has a genetically diverse, aerobic, intestinal bacterial fauna. Analysis of full-length 16S rDNA sequences from representative strains of the identified bacterial groups showed that these strains belong to KlebsieUa sp. （4N05, 4N08 and 4Nll ）, Lysinibacillus sp. （4N02）, Gamma proteobacterium （4N03）, Bacillus subtilis strain （ 4N09 ）, BrevibaciUus sp. （ 4N04, 4N06, 4N07, 4N01 and 4N10）. This study provides a theoretical basis for the microbiological control of Dendrolimu kikuchii and enriches the microbial resources library.

关 键 词：思茅松毛虫 肠道好氧细菌 多样性 ARDRA 鉴定 

分 类 号：S763.421[农业科学—森林保护学]