一直以来，基于形态学的甲藻的类鉴定都非常困难，对甲藻进行分子系统学研究，一般需要大量的甲藻纯培养物，由于淡水甲藻实验室培养非常困难，我们尝试用单细胞PCR技术扩增甲藻的某些分子标记，实验结果表明，冻融法是最适合单细胞PCR的细胞裂解方法，在单细胞PCR扩增酶系统的选择中，TakaRa Ex Taq及其配套系统具有明显的优势。从材料的保存方法来看，80%乙醇是单细胞PCR最好的材料保存方法。细胞数量并不是影响单细胞PCR结果的主导因素。
The dinoflagellates are an important group of phytoplankton in marine and fresh water, they are also a group of the dominant bloom species, and some of them even can produce toxins. Though approximately 73% -75% marine algal toxins are produced by dinoflagellates, it is seldom to find toxin production dinoflagellates in freshwater while Peridinium polonicum was the only recorded toxin production species. By observation of optical microscope and scanning electron microscopy, we firstly identified the species found in Xiangxi River, Hubei Province as Perdinium polonicum, a new record in China. Considering Perdinium polonicum’s toxin producing and water bloom forming characteristics which often causes mass mortality of fish, its impaction on environment and ecology need to be further concerned.
For a long time, morphology-based identification of dinoflagellates has always been a difficult work, and it always demands a large number of pure cultures of algae to carry out the molecular systematics research though most freshwater dinoflagellates can't be cultured in laboratory. For all of these inconveniences, we attempt to amplify some molecular markers with single-cell PCR. Our experiments show that freezing-thawing is the most suitable cell lysis method for single-cell PCR, and TakaRa Ex Taq with its supporting system has obvious advantages over others when make the decision on which amplification system to choose. As for the cell preservation, 80% ethanol is obviously the best choice in single-cell PCR. We also attempt to investigate the relations between cell amount and the result of single-cell PCR, which proved that cell amount just has slightly influence on the result of PCR in such a small cell amount.
Though single-cell PCR has many advantages, it also has very low success rate , at the same time, sorted cells can only be used once which causes the attempt to analyze more sites become impossible.So we amplified the whole genome DNA from single dinoflagellate by multiple displacement amplification and obtained large fragment, high purity and high yield products which were sufficient to meet the demands of most majority of genome analysis. In addition, we also compared the result obtained from directly single-cell PCR and reamplification of genome DNA from multiple displacement amplification. These experiments shows that both methods can achieve identical result in some chromosomal region while in others it was only firstly amplified by multiple displacement amplification cell that can get the ideal result.