we have identified species of cyanobacteria in Nakdong River using molecular genetic method, and designed primer as comparing and analyzing base sequences of 16S rRNA from environmental isolates. We compared gene amplification, cloning and base sequences with morphology, using intergenic spacer (IGS) region genes from Microcystis species and synthesized 16S rRNA. Correlation between peptide synthetase genes and the microcystin production was fast conducted for analyzing toxic species.Jong-Mun Jung, Eun-Young Jung, You-Jung Lee, Hong-Ki Park,
Mi-Eun Jung, Ki-Won Ji and Gea-Jae JooStrain Identification and Comparative Analysis of Toxigenic Cyanobacteria Determined by PCR
II. MATERIALS AND METHODS
Microcystis aeruginosa is most commonly known to produce the hepatotoxic heptapeptide microcystin in a variety of forms with varying toxicity. We compared the molecular genetic method with the morphological characteristics to strain classification of cyanobacteria in Nakdong River. We have designed PCR primers (JJM98F, JJM1141R) for cyanobacterial 16S rRNA and phycocyanin intergenic spacer (PC-IGS) gene domain. In order to confirm the production of microcystins, we have designed PCR primers for the N-methyltransferase (NMT) domain of microcystin synthetase gene mcyA and have probed 21 cyanobacteria cultures as well as several field samples.
The development of a molecular method for the identification of cyanobacteria is essential for the rapid and accurate analysis of cyanobacteria in Nakdong River. 16S rRNA and PC-IGS are currently the most promising approach for phylogenetic classification and identification of cyanobacteria18). Several previous studies of Microcystis have also shown no correlation between toxicity and other phenotypes or genotypes12,13).
In this study, NIES seven strains and environmental samples were compared with 16S rRNA and PC-IGS region, each of samples were cultured purely and identified morphology. The used primer in this study were mismatched in two bases to known 16S rRNA primer but, the result of DNA sequencing is better than that of known 16S rRNA primer. Cultured sample, 67% of the samples were identified as Microcystis aeuroginosa. As the result, cyanobacteria, Microcystis aeuroginosa may be dominant species of Nakdong River.
The differences of identification using 16S rRNA and PC-IGS were existed. If the research is performed at the many samples of cyanobacteria in involving Nakdong River, it could proceed accurately genetical classification. The data for mcyA are consistent with the observations of Neilan et al.11) and Nubel
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2)Carmichael, W.W., 1997, The cyanotoxins. In: Callow, J. A. (ED.) Advances in Botanicak research, Vol. 27, Academic press, London, 211- 256pp.
3)Ha K., H. W. Kim and G. J. Joo, 1998, The phytoplankton succession in the lower part of hypertrophic Nakdong River (Mulgum), South Korea, Hydrobiologia, 369/370, 217-227.