Development of genetic diagnosis method of toxic microalgae

有毒微藻基因诊断方法的研制

• 批准号: 13556033
• 负责人: SAKO Yoshihiko
• 金额: $ 8.58万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13556033/
• 关键词: paralytic shellfish poison; dinoflagellates; Alexandrium; molecular phylogenetic analysis; rRNA gene; bivale poisoning; poison; molecular identification; アレキサンドリウム属

Recent paralytic shellfish poisoning caused by toxic dinoflagellates which have very similar morphological features are serious problems in aquatic farms and public health. The purpose of this study are the establishment of molecular identification of toxic dinoflagellates Alexandrium spp. and development of genetic diagnosis method of toxic dinoflagellates(1) Phylogenetic analysis based on 18S rDNA sequences and the D1/D2 region in 28S rDNA were examined among the many isolates of toxic A.tamarense, A.catenella, A.tamiyavanichii, A.ostenferdii and nontoxic A.affine, A.fraterculus, A.insuetum, A.psedogonyaulax.(2) A rapid and precise identification method was established using a fluorescent, rRNA-targeted, oligonucleotide probes for toxic A.tamiyavanichii which are recently observed in Harima-Nada and the nontoxic A.affine and A.frateruculus. Each probe was species specific when applied using fluorescence in situ hybridization (FISH).(3) Real-time PCR assay was designed, evaluated and established for rapid detection and quantification of the vegetative cells and cysts (hypnozygotes) of toxic dinoflagellates A.tamarense and A.catenella.(4) The screening methods of the specific genes expressed in differential stages, such as cyst formation and germination of A.tamarense and A.catenella, were developed by use of differential display and subtractive hybridization methods. As a result, some genes specific for the sexual fusion and germination stages were detected.

近年来，由形态特征相似的有毒甲藻引起的麻痹性贝类中毒已成为水产养殖和公众健康的严重问题。本研究的目的是建立有毒甲藻亚历山大藻的分子鉴定方法。（1）利用18 S rDNA序列和28 S rDNA D1/D2区序列对塔玛甲藻、链状甲藻、塔米亚甲藻、奥斯坦菲甲藻和无毒甲藻、弗氏甲藻、猪甲藻、假刚甲藻进行了系统发育分析。(2)建立了一种快速、准确的鉴定方法，使用荧光、rRNA靶向的寡核苷酸探针，对最近在Harima-Nada中观察到的有毒的A.tamiyavanichii以及无毒的A.affine和A. frateruculus进行鉴定。当使用荧光原位杂交（FISH）应用时，每个探针是种特异性的。(3)设计、评价并建立了实时荧光定量PCR方法，用于有毒甲藻塔玛甲藻和链状甲藻的营养细胞和包囊（休眠合子）的快速检测和定量。(4)利用差异显示和差减杂交技术，建立了塔玛曲霉和链状曲霉孢囊形成和萌发等不同阶段特异表达基因的筛选方法。结果发现，一些基因特异性的有性融合和萌发阶段。

项目成果

Y.Sako, S.Hosoi-Tanabe, A.Uchida: "Fluorescence in situ hybridization using rRNA-targeted probes for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella"J.Phycol.. 40(In press). (2004)

Y.Sako、S.Hosoi-Tanabe、A.Uchida：“使用 rRNA 靶向探针进行荧光原位杂交，可简单快速地识别有毒的甲藻塔玛亚历山大藻和链状亚历山大藻”J.Phycol.. 40（正在出版）。

T.Yoshida et al.: "Purification and characterization of sulfotransferase specific to O-22 of 11-α,β-hydroxy STX from the toxic dinoflaggelate Gymnodinium catenatum (Dinophyceae)"68. 634-642 (2002)

T. Yoshida 等人：“有毒甲藻链状裸甲藻（甲藻纲）中 11-α,β-羟基 STX 的 O-22 特异性磺基转移酶的纯化和表征”68. 634-642 (2002)

S.Tanabe et al.: "Simple and rapid detection of the toxic marine dinoflagellates Alexandrium tamarense and A.catonella with fluorescence in situ hybridization (FISH) using rRNA-targeted probes"Fisheries Science. 68 Supl I. 515-518 (2002)

S.Tanabe 等人：“使用 rRNA 靶向探针通过荧光原位杂交 (FISH) 简单快速地检测有毒海洋甲藻 Alexandrium tamarense 和 A.catonella”《渔业科学》。

T.Yoshida et al.: "Geographic differences in paralytic shellfish poisoning toxin profiles among Japanese populations of Alexandrium tamarense and A.catrenella (Dinophyceae)"Phycological Research. 49. 13-21 (2001)

T.Yoshida 等人：“日本塔玛亚历山大藻和 A.catrenella（甲藻纲）种群中麻痹性贝类中毒毒素谱的地理差异”藻类学研究。

S.Tanabe et al.: "Simple and rapid detection of the toxic marine dinoflagellates Alexandrium tamarense and A.catenella with fluorescence in situ hybridization (FISH) using rRNA-targeted probes"Fisheries Sciences. 68 Supl I. 515-518 (2002)

S.Tanabe 等人：“使用 rRNA 靶向探针通过荧光原位杂交 (FISH) 简单快速地检测有毒海洋甲藻亚历山大藻和链状甲藻”《渔业科学》。