Study on DNA probe speciric for toxic dinoflagellate

有毒的链状亚历山大藻和塔玛亚历山大藻产生强烈的神经毒素，可导致沿海水域的麻痹性贝类中毒(PSP)，并造成严重的经济和公共卫生问题。然而，这些生物的形态分类仍然存在争议，因为它们的特征非常相似和可变。因此，迫切需要一种简单而客观的身份识别方法。在这项研究中，我试图通过rDNA序列来鉴定这些物种，并开发出针对每个物种的特异DNA探针。主要研究结果如下：(1)采用高效液相-荧光法对塔玛依和卡氏不动杆菌的PSP毒素进行分析，筛选出毒素含量和组成不同的典型菌株进行后续研究。(2)对这些菌株的5.8SrDNA及其侧翼内转录间隔区(ITS1和ITS2)进行扩增，并进行限制性片段长度多态性(RFLP)分析。该区域的RFLP分析有助于物种鉴别。(3)通过5.8SrDNA和ITS区的DNA序列比较进行系统发育分析，可以区分有毒和非有毒物种。(4)ITS1区3‘端(149-177bp)绝对是鉴别有毒物种的有效DNA探针。

Toxic dinoflagellates Alexandrium catenella and A.tamarense produce potent neurotoxins responsible for paralytic shellfish poisoning (PSP) in coastal waters and cause serious economic and public health problems. However, the morphological taxonomy of these organisms remains controversial because of the very similar and changeable features. Accordingly, a simple and objective method for identification is a pressing need. In this study I attempted to identify these species by rDNA sequence and to develop DNA probe specific for each species. The results are as follows ;(1) PSP toxins from many strains of A.tamarense and A.catenella were analyzed by HPLC-fluorometric analysis and the typical strains showing different toxin content and composition were selected for following studies.(2) The 5.8SrDNA and flanking internal transcribed spacer (ITS1 and ITS2) from these strains were amplified using the PCR and subjected to restriction fragment-length polymorphism (RFLP) analysis. RFLP analysis of this region is useful for species discrimination.(3) It was possible to discriminate both toxic species from non toxic ones by phylogenetic analysis using DNA sequence comparison of the 5.8SrDNA and ITS regions.(4) The 3' end of ITS1 region (149-177bp) is definitely an effective DNA probe for the identification of toxic Alexandrium species.