Analysis Of Microbiological Communities Structure Of Grass Carp Under Different Feeding Pattern By PCR-DGGE

To study and analysis the microbiological communities structure of grass carps under different feeding patterns by denaturing gradient gel.electrophoresis of PCR-amplified16S rDNA fragments (PCR-DGGE). Genome DNA was directly extracted from the samples(the intestinal wall and intestinal content of healthy grass carp, water and sediment), and then the V3region of bacterial16S rDNA gene fragments from DNA samples were amplified using the bacterial specific primers (GC-338F/534R). All the PCR products were separated by Denaturing Gradient Gel Electrophoresis (DGGE) to compare the predominant bacterial, then the singler marked bands of DGGE patterns were recovered and subcloned into E. coli respectively. Colony PCR and sequencing were applied to select the positive clones, using order arrange data to carry out same source property analysis and construct the phylogenetic tree. Then using the Quantity One software carry out diversity analysis and UPGMA analysis of the DGGE patterns. The resultant22clone sequences in16S rDNA DGGE libraries revealed that all clones show similarity in a range of89%-100%of database sequences, and among three clones present similarity up to100%. The main conclusions of the study were as follows:1. Phylogenetic analysis revealed that22clone sequences divided into5groups: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria and Cyanobacteria. It includes eleven genera.2. There exist abundant bacterial flora in water, sediment and grass carps of different feeding patterns, and each sample contains its own advantages bacteria.3. Using Quantity One software package generates similarity matrix, the UPGMA analysis result showed that the intestinal microflora of grass carp under different feeding patterns separately branch(except I1), the microflora of water and sediment gather together into a big branch. which shows that environment could affect the gut intestinal bacteria of grass carp.4. The intestine of grass carp harbors different dominant bacteria of each feeding patterns, then many common bands still exist in intestine of grass carp and it could belong to indigenous intestinal flora, which may be determined by the specificity of the host. Compared with the intestinal bacteria of the grass carp which feed fodder, the intestinal bacteria of the grass carp which feed forage contain Anoxybacillus, Anaerobacter, Ethanoligenens similar bacteria. And pattern Ⅱ, pattern Ⅲ, pattern Ⅸ contain their characteristic bacteria.