Application Of Single-cell Pcr Method, One Of Qingdao Offshore Dinophysis Genetic Characteristics

More and more molecular biological methods and techniques have been used in harmful-algal-bloom (HAB) studies on taxonomy and phylogenesis of harmful algae. The single cell PCR method, which has been developed recently, has many advantages in HAB studies, especially for those species which are difficult to culture in the laboratory. In this paper, the single cell PCR method was established in our lab and used to study the Dinophysis species collected from the sea area of Qingdao.Using the Alexandrium tamarense cultured in the laboratory, the single cell PCR method was developed and the sample preservation method was selected. The result indicated that formalin fixation (5%) method, which was often used in phytoplankton studies, was only suitable for the short-time preservation (<5days) of algae samples for single-cell PCR. The other methods, including ethanol fixation (95%), Lugol's solution preservation, or storage under -20℃could preserve the samples for a longer time (60 days in our experiment) for single cell PCR experiment.The method was used for the study of Dinophysis species collected from Qingdao. Partial sequence (D1-D2 region) of large subunit ribosomal RNA gene, and the internal transcribed spaces (ITS), were amplified and sequenced from the single Dinophysis cells. Phylogenetic studies were carried out using the sequences analyzed in this study and from the GeneBank. Based on the analytical results, the Dinophysis species from Qingdao was primarily identified as D. rotundata, which was later confirmed by morphological characteristics. Compared to the sequence information of the same species from other regions, the partial LSU sequence has 2 bp different, while the ITS sequence has 62 bp different. The LC-MS was used to analyze the dirrahetic shellfish poisoning toxins in the cells, but no OA or DTX1 was detected. This is the first study of Dinophysis using single-cell PCR method, and the ribosomal sequences of Dinophysis in China were first reported through this study. Meanwhile, the first report of D. rotundata in Qingdao indicates that the single cell PCR method is a promising method in HAB studies.