A Study On The Intestinal Bacteria Diversity Of Apostichopus Japonicus

With the rapid development of Apostichopus japonicus farming, various diseases have been in prevalent. At present, the prophylactic and sanative measures are mainly dependent on chemicals, but the misuses of antibiotic were badly for human and enviroment. The intestinal microorganism of Apostichopus japonicus has important physiological function. In order to ensure the security of Apostichopus japonicus, and find a suitable probiotics to replace antibiotics, understanding the intestinal bacteria composition of Apostichopus japonicus is very important.The traditional cultivate method is a conventional method to research microbe. In this article, three kinds of medium, including Zobell agar, Glucose (sea water) agar and TCBS agar, were used in the cultivation for screening and isolating different intestinal microflora. 16S rDNA sequencing and phylogenetic analysis showed that all of the isolates belonged to 10 generas:Pseudomonas, Vibrio, Bacillus, Shewanella, Aerococcus, Acinetobacter, Photobacterium, Staphylococcus, Pseudoalteromonas, and Kushneria. Pseudomonas was in advantage. The composition of bacteria in foregut was different from hindgut, hindgut bacteria were more plentiful than foregut both in variety and quantity, but they had the same predominant bacteria Pseudomonas. In different seasons, the intestinal bacteria composition of Apostichopus japonicus was different. In winter, Bacillus and Pseudomonas were dominant in Apostichopus japonicus gut; but in spring, the number of Bacillus appeared to be declining obviously, only Pseudomonas was in dominant.With the deepening of the research, it has been found that the great majority microbe in nature were unable to culture. The molecular biology technology, such as 16S rDNA clone library, could give an accurate analysis of the diversity of bacteria without culture. In order to construct 16S rDNA clone library of the intestinal bacteria, frist, bacterial genome kit was used to extract DNA, the concentration was 170 μg/mL.16S rDNA were amplified from the purified DNA by PCR with primer 27f and 1492r, The perfect PCR product could be obtained under the reaction condition as follows:30 cycles at 94℃ for 30 s,50℃ for 1 min, and 72℃ for 2 min. The PCR products, which were purified by the kit, were connected to pMD-18 vector with the mole ratio 1:2, then cloned into the competent cell E.coli DH5a,168 white single bacteria were obtained, the efficiency was 71%. Picking 65 single clones randomly,37 clones were positive, the efficiency of positive clone was 57%. Coverage C was 94.6%. By analysis of the restriction patterns of amplified 16S rDNA with Hae Ⅲ and Hinf Ⅰ, all positive clones were grouped into 6 operational taxonomic units (OTUs).