根据海带雄配子体抑制消减cDNA文库2个长为376 bp的表达序列标签(expressed sequence tag,EST),Blastn搜索发现它们与掌状海带的碳酸酐酶(carbonic anhydrase,CA)基因 序列(GenBank登录号:AJ130777)有70％的相似性,结合该EST以及掌状海带CA基因保守序列设计引物,扩增得到海带配子体CA基因部分开放阅读框(open reading frame,ORE)序列,再以此为基础设计基因特异性引物,然后利用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE),克隆得到一条长2804 bp的cDNA序列(GenBank登录号:JF827608),其中5′-非翻译区(untranslated region,UTR)长166 bp,3′-UTR长1765 bp,且具有明显的polyA尾巴,ORF长873 bp.它编码一个由290个氨基酸组成的前体蛋白,预测在20 Gly-21 Val处有一个典型的信号肽酶切位点,酶切后的成熟蛋白由270个氨基酸组成,具有3个锌结合的His残基所构成的催化活性中心,其分子量为30.44ku,等电点为5.06.无论在cDNA或者DNA序列上,雌、雄配子体CA基因都没有差异,且无内含子.Southem-blotting杂交结果显示,海带配子体CA基因为单拷贝基因.蛋白同源性搜索,发现该基因的编码蛋白与掌状海带的α-CA蛋白有87％的同源性.基于36个CA蛋白的氨基酸序列所构建的邻接( Neighbor-Joining)系统进化树显示,自海带配子体中所克隆的CA基因位于α-CA蛋白所组成的一支,推测它可能为α-型,因而不在线粒体中起作用.实时荧光定量PCR(quantitativereal-time PCR,Q-RT-PCR)结果表明,在增加CO2浓度的条件下,CA基因在海带雌、雄配子体中的日表达量均较未增加的条件下有所增加,由此推测该CA基因不属于胞外CA；基于海带配子体CA蛋白仅具有一个信号肽,可以推测它是定位于叶绿体外膜上,以泵的方式为叶绿体光合作用提供充足的CO2.%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 p/ 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 a-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 a-Cas,suggesting that it might be ot-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.
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