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

• Published in: Cell research Vol. 15; no. 6; pp. 465 - 473
• Main Authors: Qin, Yong Mei, Pujol, Francois Ma, Shi, Yong Hui, Feng, Jian Xun, Liu, Yi Ming, Kastaniotis, Alexander J, Hiltunen, J Kalervo, Zhu, Yu Xian
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.06.2005; National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University,Beijing, 100871, China; Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, 100871, China%Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland
• Subjects: 3-Hydroxyacyl CoA Dehydrogenases - genetics; Amino Acid Sequence; Amino acids; Biosynthesis; Centrifugation; Cloning; Cloning, Molecular; Cotton Fiber; DNA, Complementary - genetics; Endoplasmic Reticulum - enzymology; Fatty acids; Gossypium - enzymology; Leaves; Molecular Sequence Data; NADP - metabolism; Roots; Saccharomyces cerevisiae - enzymology; Sequence Alignment; Stems; Yeasts; very-long-chain fatty acids; Gossypium hirsutum; fatty acid elongation system; 3-ketoacyl-CoA reductase; short-chain alcohol dehydrogenase/reductase protein family; endoplasmic reticulum
• ISSN: 1001-0602; 1748-7838
• DOI: 10.1038/sj.cr.7290315

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 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 during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159wD mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159wD cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.