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【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】16SrDNA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。
【Objective】 The objective of this study was to investigate whether the midgut micro-organisms are related to the Bt resistance of cotton bollworm by comparing the structural and chemical constituents of the midgut bacterial community in Cry1Ac-resistant and susceptible cotton bollworm. 【Method】 The genomic DNA of the midgut of Helicoverpa armigera was first extracted and the full-length 16S rDNA fragment and V3 region were obtained by PCR. The 16S rDNA-based culture-free 16S rDNA library and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial community composition in the midgut of the Bt-resistant and susceptible strains of Bt, which is endemic to China. The bacterial community composition was analyzed and compared. 【Result】 The results of sequencing of 16S rDNA library showed that the bacterial community in the resistant and susceptible strains of Helicoverpa armigera, especially the predominant flora, were similar, but there were differences in some of the diseased colonies. The main dominant bacteria in the resistant lines were: uncultured bacterium (56.4%), Enterococcus gallinarum (17.0%) and Enterococcus casseliflavus (17.0%). The main dominant bacteria 60.2% of uncultured bacterium, Enterococcus gallinarum accounting for 19.3% and Enterococcus casseliflavus accounting for 14.7%. Subsequent PCR validation showed that some of the inferior bacteria were present in both strains. DGGE analysis showed that the similarity of the two strains of Helicoverpa armigera was 92.3%. 【Conclusion】 The results showed that the composition of gut flora of susceptible and resistant strains was very similar, suggesting that there was no direct relationship between the resistance and gut microbes.