Bt Cry8Ea蛋白对华北大黑鳃金龟幼虫(Holotrichia oblita)中肠微生物菌群的影响

摘要

Bt Cry8Ea protein presents insecticidal activity to Holotrichia oblita larvae. The interaction between Bt Cry8Ea protein and the midgut microorganism in H. oblita larvae was investigated by metagenome DNA analysis. The midguts in normal-fed or Cry8Ea protein-fed H. oblita(2nd-instar larva)were collected and then used to extract metagenome DNAs of midgut microorganism for constructing 16S rDNA gene libraries. Polymerase chain reaction restriction fragment polymorphism(PCR-RFLP)was employed to determine the polymorphisms of 16S rDNA enzyme-spliced fragments(Msp I,Rsa I,and Hae III restriction enzyme)from the midgut microorganism of two fed larvae. Different types of clones were sequenced and then aligned with NCBI gene database,and the phylogenetic tree was constructed to study the differences of microbial communities. The results showed that there were 30 bacteria in the normal group of H. oblita,and the predominant bacteria were Desulfosporosinus(29.63%),Clostridium(12.59%),and Bacillus(10.37%)respectively. However,there were only 4 bacteria in the Cry8Ea protein-fed H. oblita group,and the predominant bacteria were Enterococcus(50%)and Streptococcus(46.88%). These data revealed that the treatment with Cry8Ea protein changed the abundance and composition of midgut microbial flora.%Bt Cry8Ea蛋白对华北大黑鳃金龟幼虫有特异的杀虫活性,通过中肠微生物宏基因组分析研究了Bt Cry8Ea蛋白与华北大黑鳃金龟幼虫中肠微生物的相互作用.解剖正常饲喂和饲喂Cry8Ea蛋白的华北大黑鳃金龟Holotrichia oblita二龄幼虫中肠,提取中肠微生物宏基因组DNA,构建16S rDNA文库.利用PCR-RFLP方法分析两个处理中肠微生物16S rDNA酶切片段多态性(MspⅠ,RsaⅠ和HaeⅢ内切酶),不同克隆测序后与NCBI数据库进行对比分析,构建系统进化树,确定中肠微生物种属.结果显示,正常饲喂的华北大黑鳃金龟幼虫中肠微生物共检出30种细菌,优势菌群为变形菌门(Proteobacteria):(Desulfosporosinus,29.63%);厚壁菌门(Firmicutes):梭菌属(Clostridium,12.59%)和芽孢杆菌属(Bacillus,10.37%),而饲喂Cry8Ea蛋白的华北大黑鳃幼虫中共检出4种细菌,优势菌群为厚壁菌门(Firmicutes):肠球菌属(Enterococcus,50%)和链球菌属(Streptococcus,46.88%).结果表明,饲喂Cry8Ea蛋白后,中肠微生物的菌群结构发生了明显变化.