Fett J P, Coleman J R
Department of Botany, University of Toronto, Ontario, Canada.
Plant Physiol. 1994 Jun;105(2):707-13. doi: 10.1104/pp.105.2.707.
Two distinct cDNA clones encoding carbonic anhydrase (CA) were isolated from an Arabidopsis thaliana lambda YES library. One of these clones, CA1, encodes a 36.1-kD polypeptide and is essentially the same as a previously reported Arabidopsis CA cDNA (C.A. Raines, P.R. Horsnell, C. Holder, J.C. Lloyd [1992] Plant Mol Biol 20: 1143-1148). Comparison of the derived amino acid sequence from this clone with other plant CAs suggests the presence of a chloroplastic transit peptide, which, when cleaved, would render a mature protein of 24.3 kD. The other identified clone, CA2, encodes a 28.3-kD polypeptide, which in addition to other residue changes, is 78 amino acids shorter at the N terminus than the primary product of CA1. The two cDNAs exhibit 76.9% sequence similarity at the DNA level and 84.6% identity between the predicted amino acid sequences. A polyclonal antibody generated against pea CA (N. Majeau, J.R. Coleman [1991] Plant Physiol 100: 1077-1078) hybridized to two protein bands (25 and 28 kD) from a total leaf extract and to only one band (25 kD) from a chloroplastic protein extract. The data suggest that the CA2 protein is an extrachloroplastic form of CA, presumably localized in the cytoplasm. Southern analysis indicated that CA1 and CA2 are encoded by different genes. Northern analysis of total leaf RNA resulted in hybridization of CA1- and CA2-derived probes to two transcripts of 1.47 and 1.2 kb, respectively. These data provide additional evidence that the CA2 clone is a full-length cDNA and that two transcribed CA genes are present in the Arabidopsis genome. Transcript levels of CA1 and CA2 decreased 70 and 20%, respectively, when mature plants were transferred to dark for 24 h. Seedlings germinated in the dark showed CA1 and CA2 transcript abundance levels of 4 and 22%, respectively, when compared with light-germinated seedlings. These data suggest that expression of CA1 is light regulated and dependent of leaf and/or chloroplast development. A possible role for cytoplasmic CA in the plant cell is discussed.
从拟南芥λ YES文库中分离出两个编码碳酸酐酶（CA）的不同cDNA克隆。其中一个克隆CA1编码一种36.1-kD的多肽，与先前报道的拟南芥CA cDNA基本相同（C.A. Raines、P.R. Horsnell、C. Holder、J.C. Lloyd [1992] Plant Mol Biol 20: 1143 - 1148）。将该克隆推导的氨基酸序列与其他植物CA进行比较，表明存在一个叶绿体转运肽，切割后会产生一个24.3 kD的成熟蛋白。另一个鉴定出的克隆CA2编码一种28.3-kD的多肽，除了其他残基变化外，其N端比CA1的初级产物短78个氨基酸。这两个cDNA在DNA水平上表现出76.9%的序列相似性，预测的氨基酸序列之间的同一性为84.6%。针对豌豆CA产生的多克隆抗体（N. Majeau、J.R. Coleman [1991] Plant Physiol 100: 1077 - 1078）与总叶提取物中的两条蛋白带（25 kD和28 kD）杂交，与叶绿体蛋白提取物中的一条带（25 kD）杂交。数据表明CA2蛋白是CA的一种叶绿体以外的形式，可能定位于细胞质中。Southern分析表明CA1和CA2由不同的基因编码。对总叶RNA的Northern分析导致CA1和CA2衍生的探针分别与1.47 kb和1.2 kb的两个转录本杂交。这些数据提供了额外的证据，证明CA2克隆是一个全长cDNA，并且拟南芥基因组中存在两个转录的CA基因。当成熟植株转移到黑暗中24小时时，CA1和CA2的转录水平分别下降了70%和20%。与在光照下发芽的幼苗相比，在黑暗中发芽的幼苗中CA1和CA2转录本丰度水平分别为4%和22%。这些数据表明CA1的表达受光调节，并且依赖于叶片和/或叶绿体的发育。讨论了细胞质CA在植物细胞中的可能作用。
