利用PCR-DGGE技术分析桑天牛成虫肠道细菌菌群

利用PCR-DGGE技术分析桑天牛成虫肠道菌群结构及优势菌群,获取桑天牛肠道微生物的多样性信息。从桑天牛成虫肠道中提取细菌基因组DNA,以细菌16S rDNA基因通用引物27F/1495R和27F/519r+GC进行V3可变区PCR扩增,将长约500 bp的扩增产物经变性梯度凝胶电泳(DGGE)分离后,进行优势条带分析、DNA回收、克隆、测序等,初步得到分别属于肠杆菌属(Enterobacter)、不动杆菌属(Acinetobacter)、埃希氏菌属(Escherichia)、志贺菌属(Citrobacter)、克雷伯氏菌属(Klebsiella)、柠檬酸菌属(Shigella)、泛菌属(Pantoea)和沙雷氏菌属(Serratia)的8个细菌菌株,其中优势细菌为克雷伯氏菌属的细菌菌株,其次是沙雷氏菌属的细菌菌株。将获取的细菌16S rDNA序列在GenBank数据库中进行BLAST比对分析,相似度在97%以上的有6个菌株,其中有5个菌株与传统方法分离菌株的鉴定结果一致,表明基于16S rDNA的PCR-DGGE技术可用于桑天牛肠道菌群多样性研究。

Analysis of Intestinal Bacterial Communities in Apriona germari Adult Based on PCR-DGGE Technology

In order to acquire biodiversity information of intestinal microorganisms in Apriona germari adult,PCR-DGGE technology was employed to analyze the composition of microorganism populations and dominant bacteria in intestine of A.germari adult.After genomic DNA extraction from bacteria in intestine of A.germari adult,we amplified 16S rDNA V3 variable region by PCR with 27F/1495R and 27F/519r+GC universal primers and obtained an amplified product of around 500 bp,which was further isolated by denaturing gradient gel electrophoresis(DGGE).After dominant band analysis,DNA recovery,cloning and sequencing,8 bacterial strains were obtained.They belonged to Enterobacter,Acinetobacter,Escherichia,Citrobacter,Klebsiella,Shigella,Pantoea,Serratia respectively,among which the bacterial strain belonged to Klebsiella was the dominant strain,followed by the bacterial strain belonged to Serratia.Blast search with the obtained bacterial 16S rDNA sequences in GenBank database showed that there were 6 bacterial strains showing similarity over 97%,among which 5 bacterial strains were consistent with the classification by traditional methods.These results showed that PCR-DGGE technology based on 16S rDNA can be used to study biodiversity of intestinal microorganisms in A.germari.