Analysis of genes for calcification and photosynthesis of coccolithophorids

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1.We have isolated a partial cDNA fragment of a gene which is expressed specifically in coccolith-bearing cells of a coccolithophorid Pleurochrysis, using differential screening method. Northern analysis showed that the 2.0kb mRNA accumulated in coccolith-bearing cells, but not in naked cells. This implies that the gene could be involved in coccolith formation. Now we are trying to isolate the complete cDNA clone to analyze the structure and function of the gene product.2.To introduce genes involved in calcification into naked mutant strains, we have attmpted to develop transformation system. First, we have improved transformation by electroporation method, using Chlamydomonas as a model organism of microalgae. Highly efficient transformation (8x10^4 transformants/mug DNA,about 100 fold as efficient as the glass beads method) was achieved, by adjusting osmotic pressure of the solution for electroporation (TAP medium+40 mM sucrose) and adding cornstarch before plating. Then, conditions for screening of transformants were determined, using the coccolithophorid Pleurochrysis. Among tested regents, zeocin was the most effective to inhibit colony formation. Using the ble gene (zeocin resistant gene), conditions for electroporation of Pleurochrysis are now under investigation.3.To clarify the phylogenetic relationships of coccolithphorids, the nucleotide sequences of rbcL genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were determined from 23 species of Prymnesiophyta. Molecular phylogenetic trees were constracted using parsimony, neighbor-joining, and maximum likelihood analyzes. These trees suggest that (1) coccolithophorids from a monophyletic group, (2) the monophyletic group is consising of a clade of Emiliania, Gephyrocapsa, and Helicosphaera and a clade of other coccolithophorids, and (3) a noncoccolithophorid Isochrysis is closely related to Emiliania and Gephyrocapsa.