| In this paper, the author studied the diversity of microorganism carried by Dalian Apostichopus japonicus from the traditional microbiological cultivation and the perspective of molecular biotechnology.Through 16S rDNA sequence alignment analysis and experiments on physiological and biochemical traits, culturable bacteria on the surface of Apostichopus japonicus in different seasons were separated. The result showed that bacterial strains isolated from the surface of Apostichopus japonicus mainly including Pseudoalteromonas, Shewanella, Vibrio, Pseudomonas, Bacillus subtilis, Proteobacterium, Alteromonadaceas, Acinetobacter, Bacillus amyloiquefacieus and Staphylococcus saprophyticus. Among them, Pseudoalteromonas and Shewanella were symbiotic dominant bacterium group on the surface of Apostichopus japonicus, while Vibrio was adnascent dominant bacterium group. These three categories of bacteria all beleng to y-proteobacteria, while Pseudoalteromonas and Shewanella were allied to Alteromonadaceae phage of γ-proteobacteria. The number of bacteria separated from the surface of Apostichopus japonicus in autumn and spring was higher than that in winter, while autumn and spring was the vegetation period of Apostichopus japonicus. The number of bacteria separated from the surface of Apostichopus japonicas in their vegetation period was higher than in their non-vegetation period. It showed that the number of bacteria on the surface of Apostichopus japonicus was related to their growth status.In this paper, preliminary analysis was also done to the diversity of uncultured bacteria on the surface of Apostichopus japonicas. Molecular biotechnology such as bacteria total DNA extraction from surface of Apostichopus japonicus,16S rDNA of PCR amplification, TA cloning, positive cloning screening and restriction fragment length ploymorphism were employed. The result showed that purity of bacteria total DNA extracted by using DNA extraction kit methods was higher; 16S rDNA of PCR amplification conditions of bacteria total DNA were stated as following:predegeneration 94℃ 5 min, degeneration 94℃ 1min, annealing 50℃ 40 s, extension 72℃ 90 s and 30 cycles; Random pick up 90 transformants and 53 successful TA cloning was showed, restriction enzyme fragment Hae III and Hinf I were used for double enzyme cut of PCR amplified products and 11 different electrophoretic bands was showed, preliminary infered that contained at least 11 different species of bacteria. Using molecular biology technology in addition to cultivate separation of bacteria outside, till existed certain kinds of uncultured bacteria. |
