Cloning and functional verification of ELOVL7 gene in Eriocheir sinensis

Elongation of long-chain fatty acids (ELOVL) plays a critical role in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). To investigate the LC-PUFA biosynthesis mechanism in the Chinese mitten crab (Eriocheir sinensis), we cloned the ELOVL7 gene using rapid amplification of cDNA ends (RACE) technology. The full-length cDNA sequence was 1 600 bp, containing a 1 200 bp open reading frame (ORF) that encoded a 399-amino acid protein, with 5'-untranslated region (5'-UTR) and 3'-untranslated region (3'-UTR) lengths of 176 and 224 bp, respectively. The deduced ELOVL7 protein exhibited typical ELOVL structure, including eight transmembrane domains, multiple conserved motifs, and a histidine box (HXXHH). Amino acid sequence similarity analysis reveals high similarity between E. sinensis ELOVL7 and orthologs from the mud crab (Scylla olivacea, 73.82%) and swimming crab (Portunus trituberculatus, 73.76%). Phylogenetic tree analysis shows that E. sinensis ELOVL7 first clustered with S. olivacea and P. trituberculatus ELOVL7 to form a distinct subclade, which subsequently grouped with other crustacean ELOVL7 sequences to comprise an independent monophyletic clade. Tissue expression profiling indicates highest ELOVL7 transcript abundance in the intestine, followed by the gill. To functionally validate the enzyme, we subcloned the coding sequence of E. sinensis ELOVL7 into the pYES2 vector and transformed into Saccharomyces cerevisiae strain INVSc1, successfully establishing a heterologous yeast expression system. Heterologous expression in yeast demonstrates that ELOVL7 can catalyze the elongation of fatty acid substrate C18:1n-9 to C20:1n-9.