Cloning and Functional Analysis of Enoyl-CoA Reductase Gene BnECR from Oilseed Rape (Brassica napus L.)

Abstract

Very-long-chain fatty acids (VLCFAs) are critical components in cuticular waxes, sphingolipids, and triacylglycerols in higher plants. Biosynthesis of VLCFAs is catalyzed by the fatty acyl-CoA elongase, a membrane-bound enzymatic complex containing 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxacyl-CoA dehydratase (HCD), and trans-2,3-enoyl-CoA reductase (ECR). In this study, the primers were designed based on multiple alignments of trans-2,3-enoyl-CoA reductase (ECR) gene sequences from Arabidopsis thaliana and other plants, and the full-length cDNA, here designated BnECR, and the corresponding genomic sequences were isolated from Brassica napus using rapid amplification of cDNA ends method (GenBank accession numbers FJ899705 and FJ899705). The sequence of BnECR cDNA was 1328 bp in length excluding the poly dA tail, and the corresponding genomic sequence was 2093 bp. BnECR was composed of 4 exons and contained a 163 bp 5′ untranslated region (5′ UTR) and a 233 bp 3′ UTR. The deduced BnECR protein was 310 amino acid in length with a molecular weight of 735.78 kD and an isoelectric point of 9.52. The critical functional sites K₁₄₄ and R₁₄₅ in AtECR were unchanged in BnECR. The G₂₂₅SGGYQIPR/HG₂₃₄, which presented a non-classical NADPH-binding motif, was found in the C-terminal of BnECR. NCBI Blastn, multiple alignments and conserved domain search showed that BnECR had the highest homology to A. thaliana AtECR. RT-PCR analysis showed that BnECR was ubiquitously expressed in B. napus and preferentially expressed in the stem. The transcript level of BnECR at mid and late stages of seed development in low erucic acid rapeseed cultivar was obviously lower than that in high erucic acid rapeseed cultivar, suggesting that BnECR was involved in biosynthesis of erucic acid. The 933 bp open reading frame of BnECR was subcloned into the yeast-Escherichia coli shuttle vector pYES2.0. The recombinant plasmid was transformed into Saccharomyces cerevisiae wild type strain By4743 and the mutant strain YDL015c. With galactose as inducer, the transformant was cultured to induce the expression of BnECR. The gas chromatographic result indicated that BnECR was overexpressed effectively in S. cerevisiae, and the content of erucic acid reached 1.34% of the total fatty acid in the recombinant strain, an increase of 52% over the control. Functional complementation of BnECR in an ECR-deficient mutant yeast demonstrated that BnECR mediated the biosynthesis of VLCFAs. The results suggest that BnECR should be functional orthologue of AtECR.