Cloning and characterization of carbonic anhydrase(CA)gene from Laminaria japonica gametophytes

A suppressive subtracted cDNA library from the male gametophyte of Laminaria japonica was constructed and two 376 bp expressed sequence tags(ESTs)were screened from this library as a carbonic anhydrase gene due to their 70% identity to Laminaria digitata (GenBank accession number:AJ130777).Combined with the sequences of the ESTs and the conserved region of L. digitata,primers were designed to get part of sequences of the open reading frame(ORF)of CA gene from L. japonica.Based upon this obtained sequence,we designed gene-specific primers and cloned a full length cDNA(GenBank accession number:JF827608)of CA from L. japonica by use of rapid amplification of cDNA ends(RACE).It was composed of 2 804 bp in length,which included 166 bp 5′-untranslated region(UTR),1 765 bp 3′-UTR with a poly-A tail at its end and 873 bp ORF.The deduced precursor protein of L.japonica CA consisted of 290 amino acids,which possessed a typical signal peptide cleavage site between 20 Gly and 21 Val.The mature protein after digestion was composed of 270 amino acids,which contained a catalytic active center constituted by 3 His residues and atomic Zn.The molecular weight of the mature protein after digestion was 30.44 ku,and its pI was at 5.06.There was no difference in the sequences of CA gene either from female or from male gametophytes,and there is no intron to separate this gene.Southern-blotting analysis suggested that the CA gene had one single copy.Its homology with α-CA of L.digitata reached 87% in peptide sequence.Neighbor-Joining(NJ)phylogenetic tree inferred from 36 CA protein sequences showed that this cloned CA gene from L.japonica was clustered with other α-CAs,suggesting that it might be α-type and it couldn’t be localized in mitochondria.Quantitative real-time PCR(Q-RT-PCR)result demonstrated that the diurnal transcripts of this CA gene in the kelp gametophytes cultured under the addition of CO₂ were higher than those cultured only by agitation with filtered air at any sampling time,thus illustrating that this CA might not be extracellular.The CA might be localized on the outer envelope of chloroplasts,therefore,due to the only one signal peptide it possessed,and it could pump the chloroplasts with sufficient CO₂ for photosynthesis.