Abstract: Very-long-chain fatty acids (VLCFAs) are critical components found 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 research, 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 sequenceswere isolated from Brassica napus by using rapid amplification of cDNA ends (RACE) method (GenBank Accession No. FJ899705 and FJ899705). The sequence of BnECR cDNA was 1 328 bp (excluding the poly dA tail), and the corresponding genomic sequence was 2093 bp. BnECR was composed of four 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 MW of 735.78 kD and a pI of 9.52. The critical functional sites K₁₄₄, R₁₄₅ in AtECR were unchanged in BnECR. The G₂₂₅SGGYQIPR/HG₂₃₄ which presented a non-classical NADPH-binding motif was found in 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 middle 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 BnECRORF was subcloned into the yeast-E. coli shuttle vector pYES2.0. And then the recombinant plasmid was transformed into Saccharomyces cerevisiae wild type strain By4743 and mutant strain YDL015c, respectively. With galactose as inducer, the transformant was cultured to induce the expression of BnECR. The GC result indicated that BnECR was overexpressed effectively in S. cerevisiae, and the content of erucic acid reached to 1.34% of the total fatty acid in recombinant strain, an increase of 52% over the control. Functional complementation of BnECR in a ECR-deficient mutant yeast demonstrated that BnECR mediated the biosynthesis of VLCFAs. Our results suggest that BnECR should be functional orthologue of AtECR.

Key words: Brassica napus L, Enoyl-CoA reductase, Cloning, Erucic acid, Saccharomyces cerevisiae