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，利用紫外线和热激等物理诱变剂，对引起麻痹性贝毒的塔玛亚历山大藻(Alexandrium Tamarense)、链状亚历山大藻(A.catenella)和微小亚历山大藻(A.minuum)进行了无毒突变。因此，人们怀疑原始克隆中已经存在无毒细胞。然后，从原克隆培养物中分离出51个未经诱变剂处理的亚克隆，证实其中近一半的细胞是无毒的，这表明在长时间的分批培养中，原克隆培养物中至少有一株无毒细胞株是通过自然突变而产生的，推测无毒和有毒亚克隆中的基因组除了产毒基因外没有实质性的差异。因此，通过比较两个亚克隆的基因组，可以鉴定出该基因。我对这两个亚克隆进行了无菌培养，然后尝试按照植物的标准方法提取RNA。然而，由于塔玛曲霉的核糖核酸酶活性很高，我不能成功地完成这项工作。我优化了RNA分析的样品处理方法，目前正在使用RNA消减杂交技术对产生PSP的基因进行分析。

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.