Cloning and Analysis of the Δ~6-Fatty Acid Desaturase Gene from Nonea Capsica (Willd.) G.Don

Abstract: Delta-6 desaturased fatty acids are of major importance in animal cells as they play roles in the maintenance of membrane structure and function, in the prevention of cholesterol synthesis and as precursors of eicosanoids, including prostaglandins and leuco-trienes.γ-linolenic acid has been shown to have interesting human nutritional benefits. Fatty acid in lipid of mature seeds of Nonea capsica was analyzed by GC-MS to confirm that there is the gene of delta-6 fatty acid desaturase in Nonea capsica’s genome. Degenerate primers corresponding to the conserved histidine- rich boxes’sequences of known Echium pitardii (GenBank, AY055117) and Borago officinalish (GenBank, AF007561) were used for RT-PCR amplification from total RNA of N. capsica’s the developing seeds. The 548bp PCR product was determined as the partial sequence of delta-6 desaturase cDNA, and the sequence information coming from it was used to design two gene-specific primers for rapid amplification of cDNA ends (RACE) reactions. The full-length cDNA of the 1574bp, containing 5’and 3’ends, was obtained. Sequence analysis showed that this cDNA sequence contained an open reading frame (ORF) of 1347bp encoding 448 amino acid of 51.6KD. The deduced amino acid sequence of the ORF showed similarity to those of delta-6 fatty acid desaturases gene from other organisms, including three conserved histidine-rich boxes and two hydropathy profiles. As one of the characteristics of front-end and membrane-bound desaturases, a cytochrome b5-like domain was observed at the N-terminus. Multiple sequence alignment result showed the deduced amino acid sequence had 90.0% identity to the reported delta-6 desaturase from Borago officinalish. Phylogenetic analysis also supported that it is the nearest relative to BoD6D. Delta-6 desaturase gene cloned from N. capsica (GenBank,DQ367892) is first reported…
Key words: Nonea capsica; delta-6 desaturase; cloning; alignment; phylogeny

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