Barley Acyl Carrier Protein II: Nucleotide Sequence of cDNA Clones and Chromosomal Location of the Acl2 Gene

Abstract

ACP' is a component of plastid-located plant fatty acid synthetase. It binds acyl groups covalently via the prosthetic group, 4-phosphopantetheine, during the biosynthesis of fatty acids. Three isoforms have been identified in barley leaves (1, 3). Protein sequence data have been obtained for ACP I and 11 (3), and in addition genomic clones encoding ACP I and III have been characterized (2). Herein are presented cDNA clones for ACP II and localization of its structural gene Acl2. PCR was performed to generate ACP II encoding fragments and these were used to isolate two full-length cDNA clones (Table I, Fig. 1). Sequence comparison of the PCR products and cDNA clones (pcLH9 and pcLH 10) identified two transcripts of the Acl2 gene differing in the site of polyadenylation (Fig. 1). Multiple polyadenylation sites are known for barley ACP III and spinach ACP II transcripts (2, 6). Analysis of primary structure of ACP II reveals that (a) the transit peptide is homologous to that of barley ACP III and spinach ACP II but not to the ACP I transit peptides from the two plants (1, 5, 6), (b) at position 72 a threonine is found in the deduced protein sequence instead of the determined aspartic acid (Fig. 1), and (c) at position 100 an alanine occurs in the hitherto conserved LEEEF motif, which in barley ACP II is changed to LEEAF (Fig. 1). This substitution caused by two nucleotide changes in the codon was found in both cDNAs and in all sequenced PCR products. Southern blot analysis reveals a single hybridizing band of 4.5 kb in EcoRI digests of Betzes and Bonus barley DNA, suggesting the presence of one copy of Ac12 in the barley genome, as in the case with the Acl/ and Ac13 genes (2). No cross-hybridization of the Ac12 probe is observed with Ac/l and Ac13, which are located on EcoRI fragments larger than 10 kb (2). The 4.5 kb hybridizing band was only seen in EcoRI digests of the wheat barley disomic addition line carrying chromosome 7, and thus maps Ac12 to this chromosome. Three bands (5.5 kb, >10 kb) hybridize to the Acl2-specific probe in the Chinese Spring DNA digested with EcoRI. This suggests a sequence with homology to ACP II in each of the homologous A, B, and D wheat genomes.