对Bt蛋白敏感敏感和敏感的棉铃虫中肠细菌群落的比较

摘要：【目的】通过比较Cry1Ac蛋白抗性及敏感棉铃虫中肠细菌群落的结构组成，研究中肠微生物是否与棉铃虫Bt抗性产生有关。【方法】首先提取了棉铃虫中肠微生物基因组DNA，通过PCR扩增获得了16S rDNA全长片段及V3区。采用基于16S rDNA 的免培养技术—16S rDNA文库建立和变性梯度凝胶电泳（DGGE）研究了国内特有的Bt抗性和敏感品系棉铃虫中肠细菌群落组成，并对其进行分析和比较。【结果】16S rDNA文库测序结果表明，抗性品系与敏感品系棉铃虫中肠细菌群落特别是优势菌群非常相似，但在部分劣势菌群上存在差异。抗性品系中主要优势菌有：不可培养微生物（Uncultured bacterium）占56.4%，鹑鸡肠球菌（Enterococcus gallinarum）占17.0%，铅黄肠球菌（Enterococcus casseliflavus）占17.0%；敏感品系中主要优势菌为不可培养微生物（Uncultured bacterium）60.2%，鹑鸡肠球菌（Enterococcus gallinarum）占19.3%，铅黄肠球菌（Enterococcus casseliflavus）占14.7%。随后进行的PCR验证表明，部分有差异的劣势菌在两种品系虫体都存在。DGGE图谱分析表明，这两个品系棉铃虫中肠菌群相似性达到92.3%。【结论】敏感品系与抗性品系棉铃虫肠道菌群组成极其相似，推测抗性的产生与肠道微生物无直接关系。

Comparison of midgut bacterial community between Bt-resistant and sensitive Helicoverpa armigera

Abstract: Objective] To investigate the midgut bacterial community of sensitive and resistant Helicoverpa armigera for the emergence of resistance to Bacillus thuringiensis. Methods] Two culture-independent techniques (16S rDNA library and DGGE) were introduced. The genomic DNA of midgut bacteria was extracted. The full length gene of 16S rDNA was amplified by PCR and then cloned. The 16S rDNA library was analyzed with Alu I and Sac I. The V3 region of 16S rDNA was amplified by PCR. The intrapopulation variation and variation between resistant and sensitive Helicoverpa armigera were assayed by DGGE. Results] Analysis of 16S rDNA library showed similar patterns of midgut bacterial structure and diversity in terms of the dominant bacteria, but the inferior bacteria were significant different. The resistant population harbored abundant phylotypes belonging to uncultured bacterium (56.4%), Enterococcus gallinarum (17.0%) and Enterococcus casseliflavus (17.0%). For the sensitive population, the dominant bacteria were uncultured bacterium (60.2%), Enterococcus casseliflavus (14.7%) and Enterococcus gallinarum (19.3%). The divergence of midgut bacteria community between resistant and sensitive populations detected by 16S rDNA library analysis was verified by PCR with specific primers, and the result showed that all those inferior bacteria presented in both varieties. DGGE profile revealed that the similarity of the two varieties was 92.3%. Conclusion] The midgut bacterial community of resistant and sensitive H. armigera was similar, which had no direct effect on Bt-resistance of H. armigera.