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Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers

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Cell Research 2005年第6期15卷 465-473页

作者： Francois MA PUJOL,Alexander J KASTANIOTIS,J Kalervo HILTUNEN Biocenter Oulu and Department of Biochemistry University of Oulu P.O. Box 3000 FIN-90014 University of Oulu Finland Biocenter Oulu and Department of Biochemistry University of Oulu P.O. Box 3000 FIN-90014 University of Oulu Finland Biocenter Oulu and Department of Biochemistry University of Oulu P.O. Box 3000 FIN-90014 University of Oulu Finland

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase ***-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.

关键词： very-long-chain fatty acids endoplasmic reticulum fatty acid elongation system 3-ketoacyl-CoA reductase Gossypium hirsutum short-chain alcohol dehydrogenase/reductase protein family.

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