ARTICLE

Cell Research, 15(6):465-473, June 2005

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

Yong Mei Qin^1,2, Francois MA Pujol³, Yong Hui Shi^1,2, Jian Xun Feng^1,2, Yi Ming Liu^1,2,
Alexander J Kastaniotis³, J Kalervo Hiltunen³, Yu Xian Zhu^1,2*

¹National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, 100871, China.
²Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, 100871, China.
³Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland.

Received, Apr 5, 2005 ; Revised, May 20, 2005 ; Accepted, May 21, 2005

Correspondence: Yu Xian ZHU 86-10-6275 4427 (phone) 86-10-6275 4427 (fax) zhuyx@water.pku.edu.cn

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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.

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