ARDRA와 DGGE를 이용한 Halichondria panicea 해면의 공생세균 다양성

Abstract

Department of Biological Science and Biotechnology, Hannam University, Daejeon 34054, Republic of Korea(Received December 23, 2015; Accepted December 24, 2015)ABSTRACT: Culture-dependent ARDRA and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Halichondria panicea collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and Marine agar media. PCR amplicons of the 16S rRNA gene from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rRNA gene sequences derived from ARDRA patterns showed more than 96% similarities compared with known bacterial species, and the isolates belonged to four classes, Alphaproteobacteria , Gammaproteobacteria , Bacteroidetes , and Firmicutes , of which Alphaproteobacteria was dominant. DGGE fingerprinting of 16S rRNA genes amplified from the sponge-derived total gDNA showed 14 DGGE bands, and their sequences showed 100% similarities compared with the sequences available in GenBank. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven classes, including Alphaproteobacteria , Gammaproteobacteria , Acidobacteria , Actinobacteira , Bacteroidetes , Cyanobacteria , and Chloroflexi . According to both the ARDRA and DGGE methods, three classes, Alphaproteobacteria , Gamma-proteobacteria , and Bacteroidetes , were commonly found in H. panicea. However, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture independent method than in culture-dependent method.Key words: Halichondria panicea , 16S rRNA gene, ARDRA, bacterial diversity, DGGE, marine sponge