Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula x tremuloides.

Abstract:

:A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed. From this two different cDNA clones, denoted CA1a and CA1b, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identities around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles. Northern blot hybridization revealed a CA transcript of ca. 1300 bases, 140 bases shorter than in pea. Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography. Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme. Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants.

journal_name

Plant Mol Biol

journal_title

Plant molecular biology

authors

Larsson S,Björkbacka H,Forsman C,Samuelsson G,Olsson O

doi

10.1023/a:1005849202731

subject

Has Abstract

pub_date

1997-07-01 00:00:00

pages

583-92

issue

4

eissn

0167-4412

issn

1573-5028

journal_volume

34

pub_type

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