Characterization of a strawberry gene for auxin-binding protein, and its expression in insect cells

Abstract

A gene encoding an auxin-binding protein (ABP1) was isolated from strawberry by screening a genomic library with an ABP1 cDNA from maize. It resembles ABP1 genes from other sources both in structure (four introns) and in the high level of homology of the deduced amino acid sequence of the mature protein encoded in exons 2-5. Exon 1, encoding mainly the non-conserved signal peptide, was identified by a reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Northern analysis indicated that ABP1 transcript levels were low during fruit development, but transcripts were detected by RT PCR at all stages of receptacle swelling (auxin-dependent) and ripening (inhibited by auxin), consistent with a role for ABP1 in auxin perception. Southern blot analysis indicated a small ABP1 gene family in octoploid cultivated strawberry, and four genes were identified by comparison of genomic and cDNA sequences. RT PCR was used to amplify the complete coding region for cloning as cDNA, and a recombinant baculovirus was constructed for the expression of strawberry ABP1 in insect cells. The coding region contains three consensus glycosylation sites, and multiple bands representing a range of glycoforms of the protein were detected on western blots of insect cell extracts. Only a single band was observed in extracts of tunicamycin-treated cells, and glycosylated protein yielded a unique N-terminal amino acid sequence, allowing determination of the signal peptide cleavage site.