Characterization of &alpha type carbonic anhydrase (CA) gene and subcellular localization of &alpha A in the gametophytes of Saccharina japonica

To understand the CO2 concentration mechanism in the phaeophyte kelp Saccharina japonica, a full-length complementary DNA (cDNA) encoding carbonic anhydrase (CA) was cloned from the gametophytes based on the two screened clones from a suppressive subtracted cDNA library. The cDNA sequence was composed of 2,804 bp in length, including a 166-bp 5'-untranslated region (UTR), a 1,765-bp 3'-UTR, and an 873-bp open read frame. No intron separating this gene was found after comparing its cDNA and DNA sequences. The deduced precursor protein of S. japonica CA consisted of 290 amino acids with a typical signal peptide cleavage site between Gly 20 and Val 21 from the N terminus. The mature protein contained three conserved His residues chelated with a zinc ion constituting a catalytic active site. This cloned CA gene from S. japonica could be grouped into the alpha-type as shown by a constructed phylogenetic tree and most identity within the conserved domains characteristic of the alpha-CA. Quantitative real-time PCR results demonstrated that the diurnal transcription of this CA gene in the gametophytes cultured under the addition of CO2 or HCO3 (-) were not significantly different from those cultured only with filtered air supply at any sampling time, suggesting that this CA might not be periplasmic. After preparation of polyclonal antibody with this recombinant CA in Escherichia coli, the gold immunolocalization showed that this alpha-CA was associated with the chloroplast envelopes and thylakoid membranes suggesting that this alpha-CA could provide the chloroplasts with sufficient CO2 for carbon assimilation.