Study on DNA probe specific for toxic dinoflagellate Alexandrium

有毒甲藻亚历山大藻特异性DNA探针的研究

• 批准号: 03660192
• 负责人: SAKO Yoshihiko
• 金额: $ 1.28万
• 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: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03660192/
• 关键词: Toxic dinoflagellate; Alexandrium; Paralytic shellfish poisoning; DNA probe; Saxitoxin; Gonyautoxin; Molecular taxonomy; Monoclonal antibody; DNAプロ-ブ

Toxigenic dinoflagellates belonging to 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 and also toxin content is different among strains. Accordingly, a simple, easy and objective method for identification is a pressing need. In this study I attempted to identify both species by means of monoclonal antibodies, and to develop DNA probe specific for each species. The results are as follows;(1) Large scale cultures of A. catenella and A. tamarense with clonal and axenic conditions were established by the isolation of germinated cells from the cysts isolated from Tanabe Bay and Ofunato Bay.(2) PSP troxins of all strains were analyzed by HPLC-fluorometric analysis and then the typical strains showing different toxin content … More and composition were selected for following studies.(3) Monoclonal antibodies specific to toxic Alexandrium, which were prepared by us, could immunologically discriminate and identify inter- and intraspecies of these microalgae.(4) In toxic Alexandrium, the purification method of whole cell DNA with high molecular weight (>50kbp) was established for the first time. Amplification with two primers positioned 3'end of 17SrDNA and 5'end of 24SrDNA yielded a PCR product of 610pb in all strains of both species. The DNA fragments corresponded to the regions of ITS(internal spacer)1, 5.8S and ITS2. Digestion analysis with AluI and RsaI showed the different restriction fragment length polymorphism in both species. The identification results by the DNA fragments correspond to that of immunological analysis. From these results and also low degree of sequence similarity in the ITS regions of both species, DNA probe specific for this region is definitely an effective tool for the identification of toxic dinoflagellate Alexandrium species. Less

产毒鞭毛藻属的亚历山大菌和塔玛兰甲藻产生的强大的神经毒素负责麻痹性贝类中毒（PSP）在沿海水域，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多变，而且菌株之间的毒素含量也不同。因此，迫切需要一种简单、容易和客观的识别方法。在这项研究中，我试图通过单克隆抗体来鉴定这两个物种，并为每个物种开发特异性的DNA探针。研究结果如下：(1)从田边湾和大船渡湾分离的包囊中分离出萌发细胞，建立了大规模的克隆培养和无菌培养条件下的catenella和tamarense。(2)采用高效液相色谱-荧光法分析各菌株的PSP毒素含量，选取毒素含量不同的典型菌株进行后续研究。(3)制备的有毒亚历山Alexandrium特异性单克隆抗体能够对这些微藻进行免疫鉴别和种间及种内鉴定。(4)在有毒亚历山大菌中，首次建立了高分子量（>50kbp）全细胞DNA的纯化方法。两个引物分别位于17SrDNA的3′端和24SrDNA的5′端，扩增得到的PCR产物均为610pb。DNA片段分别对应ITS(internal spacer)1、5.8S和ITS2区域。AluI和RsaI酶切分析显示，两种植物的限制性内切片段长度多态性不同。DNA片段鉴定结果与免疫学分析结果一致。从这些结果和两种物种ITS区域的低程度序列相似性来看，针对该区域的DNA探针绝对是鉴别有毒的亚历山大属鞭毛藻物种的有效工具。少

项目成果

Y. Sako, C.H. Kim and Y. Ishida: "Mendelian inheritance of paralytic shellfish poisoning toxin in the marine dinoflagellate Alexandrium catenella" Biosci. Biotech. Biochem.56. 692-694 (1992)

Y.萨科，C.H.

S.Sawayama: "Purification and structure determination of the bacterial mating inhibitor for Chlamydomonas reinhadrtii and Alexandrium catenella." Nippon Suisan Gakkaishi. 57. 307-314 (1991)

S.Sawayama：“莱茵衣藻和链状亚历山大藻的细菌交配抑制剂的纯化和结构测定。”

Y.Sako: "Isozyme analysis of mating populations of Gonium pectorale (Chlorophyta)." J.Phycol.28. 309-315 (1991)

Y.Sako：“胸藻（绿藻）交配种群的同工酶分析。”

Y.Sako: "Mendelian inheritance of paralytic shellfish poisoning toxin in the dinoflagellate Alexandrium catenella." Biosci.Biotech.Biochem.56. 692-694 (1992)

Y.Sako：“链状亚历山大藻中麻痹性贝类中毒毒素的孟德尔遗传。”

左子 芳彦: "微生物の生態19" 学会出版センタ-, (1992)

佐子义彦：《微生物生态学19》学会出版中心，（1992）