Screening And Agarese Gene Cloning Of Agar-degrading Bacteria

The vast expanse of the ocean environment is rich in natural resources, especially the types of algae and bacteria. Seaweed is rich in many agar, and agarotic oligosaccharides can be agar-degrading bacteria degradation of output, has a wide of use. However, technical problems of manufacturing technology is a agarooligosaccharides urgent. It mainly use the enzyme hydrolysis method and chemical method, the enzymatic hydrolysis reaction of moderate consumption, with specificity and high efficiency, has become the method of preparation of agaro oligosaccharide most commonly used.Isolation of 3 strains with agar degradation function of marine agarolytic bacterium of this study from the tropical marine environment in Hainan Lingshui,Shandong Qingdao's temperate marine environment and the Gulf of Mexico agar,respectively named FG4, FG7 and FG8. The morphological characteristics were observed under the microscope and Gram staining and antibiotic resistance test were carried out. The results showed that FG7 and FG8 were both thick and red colonies. In Ampicillin, Chloramphenicol, Carbenicillin, and Streptomycin resistance experiment,Chloramphenicol and Streptomycin resistant to FG7, Chloramphenicol resistant to FG8.Identification of bacteria with universal primers 1492R and 968F identification of bacteria, the target fragment by electrophoresis, recovery of DNA fragments by connecting the transformation, select the successful cloning of attached to the carrier,then the carrier T primer M13R (-48) and M13F (-47) bacterium PCR amplification detection. The identification of the 16S rDNA target fragment sequencing, sequencing sequence by BLAST comparison and homology analysis, initially identified as FG7 is Vibrio,FG8 is Microbulbifer .In order to obtain the gene to study the function of agar degrading bacteria Vibrio sp.FG4,the full-length gene by genome walking method for Vibrio sp.FG4 amplification. Total DNA extracted from Vibrio sp.FG4, the agarase gene sequence published out its homologous gene, searching for conserved regions of the sequences of these genes and designed primers, designed specific primer for amplifying conserved region of the cloned fragment of Vibrio sp.FG4 gene conservative area in the middle, and then use the AP primers for genome walking kit and SP specific primers of Vibrio sp.FG4 genome design of genome walking, full-length gene fragment of Vibrio sp.FG4, the sequence of the cloned fragment. Sequencing resultsobtained the full-length genome of the Vibrio sp.FG4 genome, named"agaFG4-B". According to bioinformatics analysis showed that agaFG4-B gene size is 1830 bp, encoding 580 amino acids, the end of 5' the open reading frame (Open Reading Frame, ORF) a total of 6 ORF, the theoretical molecular weight of 64.490 kDa and isoelectric point of pI was 10.86.The NCBI (http://www.ncbi.nlm.nih.gov/) to construct phylogenetic tree and comparative analysis of Blastp in comparison, the results show that the "agaFG4-B"and Pseudoalteromonas sp. CY24 agarB agarase gene into the same cluster, with homology to that clone obtained "agaFG4-B" is a new type of agar enzyme gene.Based on the above that the latest technology through gene cloning, finally got 1 new agarase gene fragment Vibrio sp.FG4, not only enrich the agarolytic bacterium gene function information, and provides a theoretical basis for the future agarolytic bacteria in industrial production and application of a comprehensive understanding of the regulation of agar degrading bacteria and sub genome the subsequent expression.At the same time to promote the study of mechanisms that regulate the expression of agarolytic bacterium active substances, the development and utilization of agarolytic bacteria to produce functional oligosaccharides has created certain conditions, to speed up the application process in the industrial production.