Development and Utilization of Biosenser by Means of Genetic Marker of Harmful and Noxious Microalgae

有害和有毒微藻遗传标记生物传感器的开发和利用

• 批准号: 07456096
• 负责人: UCHIDA Aritsune
• 金额: $ 4.54万
• 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: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456096/
• 关键词: biosensor; genetic informations; monoclonal antibody; 18SrDNA; 5.8SrDNA; Alexandrium tamarense; Gymnodinium mikimotoi; Flow cytometry; Gymnodiniummikimotoi; Chattonella

Harmful and noxious microalgae in costal weaters and cause serious problems in publich health and fisheries. The morphological taxonomy of these microalgae, however, remains controversial because of the similarity and changeability of morphological features. Accordingly, a objective and precise method for identification is really reqiured. In this study we established the base of monitoring of these noxious species by flow cytometry using a monoclonal antibody and DNA probes. The results obtained are as follows ;# We enumerated the microorganisms which killed the noxious red tide phytoplankton Heterosigma akashiwo (raphidophyceae) and Gymnodinium mikimotoi, (dinophyceae) haduring their blooms which occurred in Hiroshima Bay and Uranouchi Bay or Tanabe Bay, Osaka Bay'Japan, in 1995,1996 and 1997 using the MPN method. At every sampling site in these years, the rapid increase of the algicidal microorganisms in the seawater fractions less than 0.8mum was observed at the termination period of the blooms.# We cloned and sequenced 5.8SrDNA and ITS1 / ITS2 regions of several strains of Alexandrium spp. In this experiments we it became clear that the nucleotide sequences of 3'-region of ITS1 were similar in the intratypes but not in entertypes. So, it is possible to discriminate Alexandrium tamarense and A.catenella by means of PCR methods with specific primers.# The nucleotides sequence of 18SrDNA of G.mikimotoi had 97% abd 94% similarity against Prorocentrum micans and Symbiodinium sp., respectively.# We developed a new DNA probe which is very specific for G.mikimotoi.# The G.mikimotoi cells labeled with the specific DNA brobe were easily discriminate from other microalgae by means of flowcytomete.

有害和有毒的微藻在沿海天气，造成严重的公共卫生和渔业问题。然而，由于形态特征的相似性和可变性，这些微藻的形态分类仍然存在争议。因此，确实需要一种客观和精确的识别方法。在本研究中，我们建立了利用单克隆抗体和DNA探针的流式细胞术监测这些有害物种的基础。所得结果如下：采用MPN方法，对1995年、1996年和1997年发生在日本广岛湾、天王星湾和大阪湾田边湾的有毒赤潮浮游植物赤藻异sigma akashiwo （raphidophyceae）和mikimotogymnodinium （dinophyceae）爆发期间杀死它们的微生物进行了枚举。近年来各采样点在水华终止期，0.8mum以下海水馏分中杀藻微生物数量迅速增加。#我们克隆了几株Alexandrium spp的5.8SrDNA和ITS1 / ITS2区域，并对其进行了测序。在实验中，我们发现ITS1 3'-区域的核苷酸序列在内型中是相似的，而在肠型中则不同。因此，利用特异引物的PCR方法对塔玛兰Alexandrium tamarense和A.catenella进行鉴别是可能的。# G.mikimotoi的18SrDNA序列与micans原心菌（proorocentrum micans）和Symbiodinium sp.的相似性分别为97%和94%。我们开发了一种新的DNA探针，它对g.m ikmotoi非常特殊。利用流式细胞仪可以很容易地将标记有特异DNA的G.mikimotoi细胞与其他微藻区分开来。

项目成果

左子 芳彦: "モノクローナル抗体およびリボソーマルRNA遺伝子による有毒微細藻の分子識別" 海洋. 29. 725-7312 (1997)

Yoshihiko Sako：“使用单克隆抗体和核糖体 RNA 基因对有毒微藻进行分子鉴定”Kaiyo。29. 725-7312 (1997)

A.Uchida et al.: "Enviromental and Biological Chemistry of Dimethylsulfoniopropionate and Related Sulfonium Compounds" Plenum Press New York,364 (1996)

A.Uchida 等人：“二甲基磺酰基丙酸及相关锍化合物的环境和生物化学”Plenum Press New York，364 (1996)

K. Nakanishi et al.: "Detection of the red tide-causing plankton Chattonella marina using a piezoelectric immunosensor" Analytica Chimica Acta. 325. 73-80 (1996)

K. Nakanishi 等人：“使用压电免疫传感器检测引起赤潮的浮游生物 Chattonella 码头”Analytica Chimica Acta。

Y.Sako et al.: "Phylogeneticrelationship of ciguatera-causing dinoflagellate Gambierdisucus toxicus with 18S ribosomal DNA sequence comparison" Harmful and Toxic Algal Blooms. 7. 443-446 (1996)

Y.Sako 等人：“与 18S 核糖体 DNA 序列比较引起雪卡甲藻的 Gambierdisucus 毒性的系统发育关系”有害和有毒的藻华。

T.Yoshida et al.: "Purification and properties on paralytic shellfish poisoning toxins sulfotransferase from toxic dinoflagellate Gymnodinium catenatum" Harmful and Toxic Algal Blooms. 7. 499-502 (1996)

T.Yoshida 等人：“来自有毒甲藻链状裸甲藻的麻痹性贝类中毒毒素磺基转移酶的纯化和特性”有害和有毒的藻华。