Analysis Of Bacterial Diversity In The Intestine Of Apostichopus Japonicus By 16S RDNA

In this study, we investigated the intestinal bacterial diversity of A. japonicus using 16S ribosomal DNA gene (rDNA) clone library and Polymerase Chain Reaction/Denaturing Gradient Gel Electrophoresis (PCR-DGGE).Constructing the 16S rDNA clone library, as many as 470 positive clones of 16S rDNA clone library were selected and digested with the restriction endonucleases HinfⅠ and Hae Ⅲ. The patterns of the bands were then used to classify clones into operational taxonomic units (OTUs), and 188 of individual purified PCR products were selected for sequencing and classified into 106 operational taxonomy units (OTUs). The coverage of the library was 77.4%. Eight major phylogenetic lineages were identified:y-Proteobacteria (76.4%), a-Proteobacteria (6.6%), Firmicute (6.6%), ε-Proteobacteria (5.7%), Actinobacteria (1.9%), 8-Proteobacteria (0.9%), Fusobacteria (0.9%) and Verrucomicrobia (0.9%). Approximately 88.7% of the sequences were affiliated with Proteobacteria, with y-Proteobacteria being the predominant group, comprising 81 individual OTUs. The intestinal bacteria of A. japonicus belonged to 22 genus, they were Arcobacter, Vibrio, Psychrobacter, Pseudomonas, Bacillus, Photobacterium, Francisella, Legionella, Acinetobacter, Acanthamoeba, Staphylococcus, Brachybacterium, Pseudoalteromonas, Streptomyces, Shewanella, Aerococcus, Holosporaceae bacterium, uncultured bacterium, Aliivibrio, Thalassomonas, Neptunomonas and Fangia hongkongensis, with Vibrio being the predominant bacteria.Using Polymerase Chain Reaction/Denaturing Gradient Gel Electrophoresis (PCR-DGGE) investigated the intestinal predominant bacterial of A. japonicus, the V3 regions of the amplified 16S rDNA sequences were subjected to PCR-DGGE.16 major bands in the DGGE profiles were eluted from the polyacrylamide gels, cloned, and sequenced for each band. Taken together, the results indicated that Pseudoalteromonas, Vibrio, Propionigenium, Pseudomonas, Shewanella, Leisingera, Arcobacter, Alistipes, Ferrimonas and Rhodobacteraceae were the predominant bacteria that live in the intestine of A. japonicus. DGGE profiles and sequence analyses revealed Vibrio as the predominant bacteria in the intestine of A. japonicus.These results suggested that 16S rDNA clone library and PCR-DGGE analysis were effective techniques for monitoring and analyzing the intestinal bacterial diversity of A. japonicus, providing vital information that could be used to provide better farming for the aquaculture of A. japonicus. Both methods identified Proteobacteria as the major flora and Vibiro as the predominant bacteria in the intestine of A. japonicus.