Study on DNA probe speciric for toxic dinoflagellate

有毒甲藻特异性DNA探针的研究

• 批准号: 05660208
• 负责人: SAKO Yoshihiko
• 金额: $ 1.41万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660208/
• 关键词: Toxic dinoflagellate; Alexandrium; Paralytic shellfish poisoning; Saxitoxin; Gonyautoxin; Molecular identification; DNA probe

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.

有毒的甲藻Alexandiumcatenella和A.tamarense在沿海沃茨产生导致麻痹性贝类中毒（PSP）的强效神经毒素，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，因为非常相似和多变的特征。因此，迫切需要一种简单而客观的识别方法。在本研究中，我试图通过rDNA序列鉴定这些物种，并开发针对每个物种的DNA探针。主要研究结果如下：（1）采用高效液相色谱-荧光分析法对塔玛曲霉和链状曲霉的PSP毒素进行了分析，并选取了毒素含量和组成不同的典型菌株进行了研究。(2)利用PCR扩增了这些菌株的5.8SrDNA和侧翼内转录间隔区（ITS 1和ITS 2），并进行了限制性片段长度多态性（RFLP）分析。该区域的RFLP分析可用于物种鉴别。(3)利用5.8SrDNA和ITS区的DNA序列进行系统发育分析，可以区分这两种有毒的物种和无毒的。(4)ITS 1区的3'端（149- 177 bp）是鉴定有毒亚历山大藻的有效DNA探针。

项目成果

Y.Sako: "Studies on stability and heredity of PSP toxin composition in the toxic dinoflagellate Alexandrium" J.Phycol. (投稿予定).

Y.Sako：“有毒甲藻亚历山大藻中 PSP 毒素成分的稳定性和遗传性研究”J.Phycol（待提交）。

C.-H.Kim: "Variation of toxin production and compostition in axenic cultures of Alexandrium catenella and A.tamarense" Nippon Suisan Gakkaishi. 59. 633-639 (1993)

C.-H.Kim：“链状亚历山大藻和 A.tamarense 无菌培养物中毒素产生和成分的变化”Nippon Suisan Gakkaishi。

M.Adachi: "Cross-reactivity of five monoclonal antibodies to various isolates of Alexandrium as determined by anindirectimmunofluorescencemethod" Nippon Suisan Gakkaishi. 59. 1807- (1993)

M.Adachi：“通过间接免疫荧光法测定五种单克隆抗体对不同亚历山大藻分离株的交叉反应性”Nippon Suisan Gakkaishi。

M.Adachi: "The identification of conspecific dinoflagellates Alexandriun tamarense from Japan and Thailand by monoclonal antibodies." Nippon Suisan Gakkaishi. 59. 327-332 (1993)

M.Adachi：“通过单克隆抗体鉴定来自日本和泰国的同种甲藻 Alexandriun tamarense。”

Y.Sako: "Toxic Marine Phytoplankton" Elsevier(in press), (1995)

Y.Sako：“有毒海洋浮游植物”爱思唯尔（印刷中），（1995 年）