| Research Abstract |
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.
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