Cloning and sequencing of cDNA for glycolate oxidase from pumpkin cotyledons and northern blot analysis

Abstract

A cDNA clone for glycolate oxidase (EC 1.1.3.1) was isolated by an immunochemical method from a cDNA expression library constructed from poly(A)+-RNA of green pumpkin cotyledons. The analysis of in vitro transcription-translation products of the cDNA insert revealed that the cDNA clone contained the complete coding region for glycolate oxidase. The entire insert of the cDNA was 1,440 nucleotides in length and encoded 367 amino acid residues, equivalent to a molecular mass of 40,353 daltons. The amino acid sequence of the C-terminal tripeptide was Pro-Arg-Leu, which is slightly different from the proposed signal for targeting to microbodies, Ser-Lys/Arg/His-Leu. Characteristic hydrophilic domains observed in the C-terminal regions of most microbody proteins were found in the deduced sequence of glycolate oxidase by hydropathy analysis. Immunoblot analysis showed that the amount of glycolate oxidase was low in dark-grown cotyledons and increased during greening of pumpkin cotyledons. Northern blot analysis showed that the probe could hybridize with a single 1.5-kb species of mRNA from pumpkin cotyledons and that the amount of the hybridizable mRNA increased dramatically during greening of the cotyledons. This observation indicates that the induction of glycolate oxidase during greening of the cotyledons is due to an increase in the level of the mRNA.