Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco

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Abstract
Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme as two closely related polypeptides of 44 and 42.5 kDa from tobacco stems. In this paper, we report partial amino acid sequences of these two polypeptides. Based on the peptide sequences mixed oligonucleotides were used to screen a tobacco stem cDNA library and CAD cDNA clones encoding the two polypeptides were identified. DNA sequence comparisons indicate very high sequence identity between these clones both in the coding and in the 5′ and 3′ untranslated sequences. The close similarity between the two CAD genes leads us to suggest that they do not represent different isoforms but are the same gene from each of the two parental lines of Nicotiana tabacum cv. Samsun. Sequence comparisons with alcohol dehydrogenase 1 (ADH1) from yeast shows sequence similarities of ca. 30%, while comparisons with maize, barley and potato ADH1 sequences show similarities of not more than 23%.