家蚕幼虫中肠细菌群落多样性的PCR-DGGE和16S rDNA文库序列分析

Bacterial community in midguts of the silkworm larvae estimated by PCR/DGGE and 16S rDNA gene library analysis

采用基于16S rDNA的变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和16S rDNA文库序列分析的手段,研究了重要经济昆虫家蚕Bombyxmori2个品系——专食性品系C108和广食性品系SCN2幼虫中肠内的细菌群落多样性,同时还探讨了食料对家蚕中肠内细菌群落结构的影响。文库序列分析表明,PCR扩增得到的16S rDNA基因代表了家蚕中肠内的41种细菌系统发育型(phylotype),大多数属于Proteobacteria,其次是Lactobacillales。此外,还有少数属于Deinococcus-Thermus、Bacillales、Clostridiales和Actinobacteria,尚有5种系统发育型不能确定其所属类型。家蚕的这2个品系中,肠球菌属Enterococcus是其中肠细菌的优势菌群,栖热菌属Thermus是次优势菌群。优势菌肠球菌属的组成在品系和不同食料喂养条件下有着一定的变化,无桑饲料喂养条件下SCN2品系中肠内还出现了新的次优势菌葡萄球菌(Staphylococcus)。DGGE图谱显示家蚕低龄幼虫和高龄幼虫肠道细菌格局存在差异,推测可能与其发育期生理状态的差异有关。本研究结果提示家蚕肠道特殊菌群的出现可能与其特殊的食性有一定的关系,食料改变、生长受阻后肠道微生态平衡也发生变化。

We inventoried the bacteria community and the effects of diet on bacterial composition in larval midguts of two strains （the monophagous C108 and the polyphagous SCN2） of the silkworm, Bombyx mori L., an economically important insect, using 16S PCR-DGGE and clone libraries. DNA sequence analysis indicated that the PCR-amplified 16S rDNA genes represented 41 phylotypes of bacteria. Most of them belonged to the Proteobacteria and Lactobacillales, whereas less dominant taxa including members of the Deinococcus- Thermus, Bacillales, Clostridiales and Actinobacteria were also found in the midgut bacterial community. Phylotypes of Enterococcus were the most dominant group in both strains tested, followed by Thermus. Bacterial composition or population structure of Enterococcus varied between the two strains and in the same strain fed with different diets. Especially, when strain SCN2 was fed with mulberry-free artificial diet, a midgut phylotype belonging to Staphylococcus appeared with a fairly high proportion. DGGE profiles demonstrated that there were differences in the bacterial pattern between young and old larvae. This might be related with the differences of physiology between the two developmental stages. The results suggested that the appearance of special bacteria might associate with the special phagy of the silkworm, and unfit diet might destroy the balance of midgut microbiota to block normal growth of the silkworm.