桑粒肩天牛幼虫肠道微生物多样性的分子生物学方法分析-molecular biological analysis of intestinal microbial diversity of apriona germari larvae.docxVIP

重庆大 重庆大学硕士学位论文 英文摘要 PAGE PAGE VI ABSTRACT Apriona germari (Hope) is an important pest of many kinds of trees, and the destructive pests distribute almost all over the world. Researches have showed that intestinal bacteria have very close relationship to the well-being of the insect host in many aspects, e.g. nutrient and metabolic activities, and especially immune functions. A substantial part of intestinal bacteria of this beetle are still to be thoroughly described for the reason that less than 1% of the microbes in natural environments could be successfully isolated by culture-dependent techniques under laboratory conditions. Fortunately, in the last decades, several 16S rDNA-based molecular methods have been developed for bacteria identification. The gut flora of A. germari larvae was investigated by three 16S rDNA-based molecular approaches to provide novel insights into the uncultured microbes in guts and help attempt to control the pest. These methods were denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP) analysis of traditional 16S rDNA clone library and directly randomly sequencing of normalization 16S rDNA clone library. DGGE analysis inferred 22 different bacteria from 15 genera by sequencing principal bands and an uncultured Proteobacterium and a Klebsiella appeared in every sample. Band E and L identified as Bacillus sp. and an uncultured Klebsiella sp. were the strongest bands. DGGE band patterns showed that gut flora in summer was more complex than in winter generally. A traditional 16S rDNA clone library and a normalization bacteria 16S rDNA clone library were established. Restriction fragment length polymorphism (RFLP) analysis of traditional clone library clustered tested 175 clones into 48 different groups, and the available sequences representing each RFLP-type were closely related to 48 different bacteria in 22 genera. The predominant bacteria were species in genera Klebsiella, Lactococcus and Ent