Studys On The Uncultured Microorganism Flora From Apostichopus Japonicus Surface And The Antagonistic Bacterium

According to the statistics,the aquaculture value of Apostichopus japonicus in Liaoning Province has accounted for more than one-third of China in 2010.It has been the economic pillars of Liaoning Province and the sunshine industry of China.However,there was bacteria contamination phenomenon in the farming process of Apostichopus japonicus.The information of Apostichopus japonicus microbial composition will not only being a theoretical basis for the study on Apostichopus japonicus pathogens but also be good for discovering new microbe resources and metabolic products.In order to construct 16SrDNA clone library of the bacterium from Apostichopus japonicus surface,bacterial genome kit was used to extract total DNA of bacterium from Apostichopus japonicus surface.Firstly,16SrDNA 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:95 ? for 5 min;30 cycles at 95 ? for 40 s,55 ? for 1 min,72 ? for 2 min;72 ? for 10 min.The concentration of recovery fragment was 100 pg/mL.The PCR products which purified by the kit were connected to pMD19-T vector with the mole ratio 1:5.They were cloned into the competent cell E.coli DH5a,225 white single clones were obtained.Picking 72 single clones randomly,34 clones were positive.The efficiency of positive clone was 47%.Coverage C was 92.5%.By analyzing the restriction patterns of amplified 16SrDNA by Hae?and Hinf ?,all positive clones were grouped into 7 operational taxonomic units(OTUs).Four potential unculturable microorganisms were found by comparition with the restriction fragment length polymorphism analysis of cultured microo1ganisms from Apostichopus japonicus surface.Antibiotics were often used to inhibit and treat the bacterial diseases of Apostichopus japonicus.Although these medicaments could inhibit or kill pathogens,they decompose ecological balance of normal microorganisms in the breeding environment.Antagonistic bacterium not only can reduce farming costs but also boost the safety and health of the Apostichopus japonicus products by inhibiting the growth of pathogens.Antagonistic bacterium were screened from the microorganisms isolated from the surface and the intestinal of Apostichopus japonicus.The results of filter paper method were 11 antagonistic bacterium against Edwardsiella tarda,Et,7 antagonistic bacterium against Vibrio parahemolyticus,8 antagonistic bacterium against Vibrio splendidus,14 antagonistic bacterium against ZZ-14 and3 antagonistic bacterium against CT-7.The results of cross-streaking method were 6 antagonistic bacterium against Edwardsiella tarda,Et,20 antagonistic bacterium against Vibrio parahemolyticus,5 antagonistic bacterium against ZZ-14 and4 antagonistic bacterium against CT-7.Antagonistic mechanisms were studied for the 11 antagonistic bacterium against Edwardsiella tarda,Et and the 7 antagonistic bacterium against Vibrio splendidus.Conclusions were as follows:Antagonistic mechanisms of one antagonistic bacteria were competing nutrients and living space in the antagonistic bacterium against Edwardsiella tarda,Et.Four antagonistic bacterium produced antibacterial substances.One antagonistic bacteria competed nutrients and living space and produced antibacterial substances.Antibacterial substances were polar molecules.Bacteria CG-2-1 produced ?-1,3-glucanase.Antagonistic mechanisms of the7 antagonistic bacterium against Vibrio splendidus were competing nutrients and living space and producing antibacterial substances.Antibacterial substances were polar molecules.All of the 7 antagonistic bacterium didn't produce ?-1,3-glucanase?chintinase and cellulase.