Studies on the mechanism of the toxin production in Alexandrium spp.

亚历山大藻产毒机制的研究。

基本信息

批准号：
11660182
负责人：
ISHIMARU Takashi
金额：
$ 0.58万
依托单位：
Tokyo University of Fisheries (TUF)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660182/
关键词：
Paralytic Shellfish Poisons Alexandrium Toxin production 連続培養

项目摘要

In order to identify the gene concernig the production of paralytic shellfish poisoning (PSP), I tried to make non-toxic mutants of PSP causative dinoflagellates Alexandrium tamarense, A.catenella and A.minutum by using physical-mutagens such as UV and heat-shock.In the sub-clones of A.tamarense isolated by micoropipet method after the treatment by UV radiation, non-toxic sub-clones appeared in a very high percentage (78%). Therefore, it was suspected that non-toxic cells had already existed in the original clone. Then, 51 sub-clones were isolated without mutagen treatment from the original clone culture, and it was confirmed that almost a half of the cells in that were non-toxic suggesting that at least a single non-toxic cell strain occurred in the original clone culture by a natural mutation in the long duration of batch culture.It is presumed that there is not a substantial difference between the genomes in the non-toxic and toxic sub-clones except in the toxin-producing gene. Thus, by comparing the genomes of the both sub-clones the gene could be identified.I made axenic cultures for the both sub-clones and then tried to extract RNA according to the standard method for plant. However, I could not succeed in it because of very high activity of RNase in A.tamarense. I optimized the sample processing method for the RNA analysis and, currently, the analyses of the PSP-producing gene by using subtractive hybridization of RNA are ongoing.

为了鉴定与麻痹性贝类中毒（PSP）产生相关的基因，我尝试通过使用物理诱变剂（例如紫外线和热）来制备PSP致病甲藻亚历山大藻、链状甲藻和微小甲藻的无毒突变体。微管法分离的塔玛亚克隆经紫外辐射处理后，出现了无毒的亚克隆。高比例（78%）。因此，人们怀疑原始克隆体内已经存在无毒细胞。然后，从原始克隆培养物中分离出51个未经诱变剂处理的亚克隆，并证实其中几乎一半的细胞是无毒的，这表明原始克隆中至少存在单一无毒细胞株推测无毒亚克隆和有毒亚克隆的基因组除了产生毒素的基因外没有实质性差异。因此，通过比较两个亚克隆的基因组，可以鉴定该基因。我对两个亚克隆进行了无菌培养，然后尝试根据植物的标准方法提取RNA。然而，由于 A.tamarense 中的 RNase 活性非常高，我未能成功。我优化了RNA分析的样品处理方法，目前正在使用RNA消减杂交对PSP产生基因进行分析。