TAILIEUCHUNG - Báo cáo Y học: Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution

Genomicfattyacid elongation 1 (FAE1) clones from high erucic acid (HEA)Brassica napus, Brassica rapaandBras-sicaoleracea, and lowerucicacid (LEA)B. , were amplifiedbyPCRand expressed inyeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing theFAE1genes from HEA Brassicaspp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing theFAE1gene from LEA B. napuswere identical to control yeast samples | Eur. J. Biochem. 269 5625-5631 2002 FEBS 2002 doi Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution Vesna Katavic1 Elzbieta Mietkiewska2 3 Dennis L. Barton1 E. Michael Giblin2 Darwin W. Reed2 and David C. Taylor2 1 Saskatchewan Wheat Pool Agricultural Research and Development Saskatoon Canada 2National Research Council of Canada Plant Biotechnology hisittute Saskatoon Canada 3Plant Breeding and Acclimatization Institute Mlochow Research Center Poland Genomic fatty acdl etongstton 1 FAE1 clones from high erucic acid HEA Brassica napus Brassica rapa and Brassica oleracea and low erucic acid LEA B. napus cv. Westar were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenyl alanine in LEA B. napus cv. Westar. Using site directed mutagenesis the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids VLCMFAs indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus for the first time the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the .

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