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

为了鉴定麻痹性贝毒（PSP）产生的相关基因，采用紫外线（UV）和热激等物理诱变剂，对引起PSP的塔玛Alexandriumtamarense、链状甲藻A.catenella和微小甲藻A.minutum进行了无毒突变体的筛选，结果表明，经紫外线处理后，用微量吸管法分离的塔玛甲藻亚克隆中，无毒亚克隆的比例很高（78%）。因此，人们怀疑在原始克隆中已经存在无毒细胞。然后，从原始克隆培养物中分离出51个亚克隆，并且证实其中几乎一半的细胞是无毒的，这表明在原始克隆培养物中通过长期分批培养的自然突变至少出现了单个无毒细胞株。毒性和毒性亚克隆，除了产生毒素的基因。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，然后尝试按照植物标准方法提取RNA。然而，我不能成功，因为非常高的活性RNase在A.塔玛。我优化了用于RNA分析的样品处理方法，目前正在使用RNA的消减杂交分析PSP产生基因。

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.