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 ofintestinal aerobic bacteria from 4th instar larva of
孙佑赫 熊智 王金华 张武先 王海林
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作者单位：西南林业大学昆明650224
中文关键词：思茅松毛虫，肠道好氧细菌，多样性，ARDRA，鉴定
英文关键词：Dendrolimu kikuchii，intestinal aerobic bacteria，diversity，ARDRA
中文摘要：从思茅松毛虫Dendrolimu kikuchii Matsumura 4 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16S rDNA 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( Klebsiella sp． )，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 brevis strain)，后2 株是沼泽短芽孢杆菌(Brevibacillus limnophilus strain)，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。
英文摘要：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 ofintestinal aerobic bacteria was established using the PCR technique ( primer27f，1492r) and ARDRA patterns，whichused enzyme digestion (HaeⅢ and HindⅢ，HinfⅠ and TaqⅠ) of cloned 16S rDNA gene sequences． The results showthat 11 strains could be divided into 6 groups at a similarity level of 84% ． The results prove that Dendrolimu kikuchii hasa genetically diverse，aerobic，intestinal bacterial fauna． Analysis of full-length 16S rDNA sequences from representativestrains of the identified bacterial groups showed that these strains belong to Klebsiella sp． ( 4N05，4N08 and 4N11 )，Lysinibacillus sp． (4N02)，Gamma proteobacterium (4N03)，Bacillus subtilis strain (4N09)，Brevibacillus sp． (4N04，4N06，4N07，4N01 and 4N10 ) ． This study provides a theoretical basis for the microbiological control ofDendrolimu
kikuchii and enriches the microbial resources library．，identification.