Cloning And Characterization Of 18S,28S And 5.8S Ribosomal RNA Genes And Internal Transcribed Spacers Of Seven Species/strains In Genus Prorocentrum

The ribosomal RNA genes (rDNA, including the 18S rDNA, partial of the 28S rDNA and 5.8S rDNA) and the ITS regions of seven species/strains of Prorocentrum were cloned and sequenced. Corresponding sequences of several other species/strains of Prorocentrum were obtained from GenBank. Then the phylogenetic trees of Prorocentrum were constructed with the methods of NJ (Neighbour-Joining) and ME (Minimum Evolution) . And the sequences of the species/strains were analyzed in order to find out the highly variable region to design the species-specific probes.The results indicated that there were 315 variable sites in the 1767 18S rDNA sites of 17 strains of Prorocentrum, and the average distance(Kimura-2-parameter distance) of the species/strains was 0.049. The sequence distances between the seven species/strains of Prorocentrum in this study were very little, but we do find some species-specific sequences in the gene.In the partial sequence of 28S rDNA, there were obvious variable regions and conservative regions. Sequences in the variable regions were identical in the same species but quite different from other species, which is very useful for the design of the species-specific probes.There was a little variation between the different strains of the same species in the ITS1-5.8S-ITS2 region, while the variations between the species were much distinct. Thus it was also thought to be an excellent region to design probes.The comparison of DNA sequences of the several regions of the seven species/strains showed that the variation between the 28S rDNA partical sequences and ITS region was as much as about 12 to 36 times the variation between the 18S rDNA. And the 28S rDNA partical sequences and ITS regions are adapt to the molecular phylogenetic analysis of Genus Prorocentrum.The study on the Promcentrum sp which redulted in the red tide in the East China Sea and P. dentatum (CCMP1517) indicated that they had the identical sequence in 18S rDNA and had only one different base both in ITS1-5.8S rDNA DTS2 region and the partial sequence of the 28S rDNA. Acording to the sequences they were presumed to be the same species.The P. micans strain separated from the South China Sea was much different from the strain separated from American coast in 28S rDNA and ITS sequence, and obvious difference could be seen under the scaned electron microscope. Further study needs be done by taxonomist to reveal the true relationship between them.