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

doi:10.13560/j.cnki.biotech.bull.1985.2017-0236

生物技术通报 ›› 2017, Vol. 33 ›› Issue (8): 126-131.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0236

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

王伟¹, 赵丹¹, 郭巍^1，2, 李新娜³, 张雅昆¹, 闫晓平¹

1. 河北农业大学植物保护学院河北省农作物病虫害生物防治工程技术中心，保定 071001；
2. 北京农学院植物科学技术学院，北京 102206；
3. 邢台市农业科学研究院，邢台 054000

收稿日期:2017-03-25 出版日期:2017-08-01 发布日期:2017-08-01
作者简介:王伟，男，博士研究生，研究方向：生防微生物与基因工程；E-mail：wwjiayou85@126.com
基金资助:
国家现代农业产业技术体系项目（CARS-14）

Effects of Bt Cry8Ea Protein on the Midgut Microbial Flora of Holotrichia oblita Larvae

WANG Wei¹, ZHAO Dan¹, GUO Wei^1，2, LI Xin-na³, ZHANG Ya-kun¹, YAN Xiao-ping¹

1. College of Plant Protection，Agricultural University of Hebei，Baoding 071001；
2. College of Plant Science and Technology，Beijing University of Agriculture，Beijing 102206；
3. Xingtai Academy of Agricultural Sciences，Xingtai 054000

Received:2017-03-25 Published:2017-08-01 Online:2017-08-01

摘要/Abstract

摘要： 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Hae Ⅲ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。

关键词: 华北大黑鳃金龟, 中肠微生物, Bt Cry8Ea蛋白, PCR-RFLP

Abstract: 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.

Key words: Holotrichia oblita, midgut microorganism, Bt Cry8Ea protein, PCR-RFLP

王伟, 赵丹, 郭巍, 李新娜, 张雅昆, 闫晓平. 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[J]. 生物技术通报, 2017, 33(8): 126-131.

WANG Wei, ZHAO Dan, GUO Wei, LI Xin-na, ZHANG Ya-kun, YAN Xiao-ping. Effects of Bt Cry8Ea Protein on the Midgut Microbial Flora of Holotrichia oblita Larvae[J]. Biotechnology Bulletin, 2017, 33(8): 126-131.

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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

Effects of Bt Cry8Ea Protein on the Midgut Microbial Flora of Holotrichia oblita Larvae

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