Volume 15, No 6, Jun 2005
ISSN: 1001-0602
EISSN: 1748-7838 2018
impact factor 17.848*
(Clarivate Analytics, 2019)
Volume 15 Issue 6, June 2005: 465-473
ORIGINAL ARTICLES
Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers
Yong Mei Qin1,2, Francois MA Pujol3, Yong Hui Shi1,2, Jian Xun Feng1,2, Yi Ming Liu1,2, Alexander J Kastaniotis3, J Kalervo Hiltunen3, Yu Xian Zhu1,2*
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.
Correspondence: Yu Xian ZHU(zhuyx@water.pku.edu.cn)
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.
FULL TEXT | PDF
Browse 1937
