Nucleotide sequence of a cDNA clone encoding a precursor to stearoyl-(acyl-carrier-protein) desaturase from spinach, Spinacia oleracea.

Abstract

Stearoyl-(acyl-carrier-protein) desaturase (SAD; EC 1.14.99.6) introduces the first double bond into C18 fatty acids in higher plants and, therefore, it is involved in controlling the extent of unsaturation of membrane lipids [2]. We report here the isolation and characterization of a cDNA clone that encodes a precursor to SAD of spinach (Spinacia oleracea cv. Viroflay). A cDNA library constructed in 2ZapII from spinach cotyledons was probed with a 1.2 kb fragment of DNA, which was obtained by the polymerase chain reaction in which total cDNA from castor bean leaves and homologous primers for castor bean SAD cDNA [5], namely 5'-CTCAATCCTTTCCTTTCTC-3' and 5'-CTACAGCTTCACTTGCCTA-3' , were used. The nucleotide sequence of the cloned cDNA of spinach SAD was determined by a modified version of the dideoxy chain termination method [4]. The nucleotide sequence encoding the spinach SAD along with its deduced amino acid sequence is shown in Fig. 1. The cDNA clone contained an insert of 1610 bp with a 1097 bp open reading frame. The sequence flanking the first methionine codon at base 30 is an exact copy of the proposed consensus sequence for initiation of translation in plants, AACAAUGGC [3]. The open-reading frame encodes a polypeptide of 399 amino acid residues, a number close to that deduced for the amino acid residues of precursors to SAD in castor bean [5], cucumber [5] and safflower [1], namely 396. By analogy to the safflower SAD [5], it is predicted that a transit peptide of 3 S amino acids is cleaved from the spinach precursor to produce a mature protein of 364 amino acids. The extent of homology between the deduced amino acid sequence of the precursor to spinach SAD and those of precursors to SAD from castor bean, cucumber and safflower is 84~o, 81~o and 82~o, respectively.