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

为了鉴定麻痹性贝类中毒（麻痹性贝类中毒）产生的相关基因，我尝试利用紫外线和热休克等物理诱变剂，制备麻痹性贝类中毒致病鞭毛藻tamarense Alexandrium、catenella a.m utum a.m utum无毒突变体。紫外辐射处理后用微吸管法分离的塔玛氏A.tamarense亚克隆中，无毒亚克隆的比例很高（78%）。因此，怀疑在最初的克隆中已经存在无毒细胞。然后，从原始克隆培养中分离出51个亚克隆，未经诱变剂处理，证实其中几乎一半的细胞是无毒的，这表明在长时间的批量培养中，至少有一个无毒细胞株是通过自然突变在原始克隆培养中产生的。据推测，除了产毒基因外，无毒亚克隆和有毒亚克隆的基因组之间没有实质性差异。因此，通过比较两个亚克隆的基因组，可以鉴定出该基因。我对两个亚克隆进行了无性系培养，然后按照植物的标准方法尝试提取RNA。然而，由于A.tamarense的RNase活性非常高，所以我没有成功。我优化了RNA分析的样品处理方法，目前正在进行RNA减法杂交分析产生psp的基因。

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.