Molecular cloning of full-length cDNA encoding delta-9 desaturase through PCR strategies and its genomic organization and expression in grass carp (Ctenopharyngodon idella).

Abstract

Desaturases are enzymes that catalyze double bond formation in fatty acids, which is a critical step in the synthesis of unsaturated fatty acids in organisms. Desaturase cDNA has been cloned from various species. Here we report the cloning of a full-length cDNA of Delta(9)-desaturase from grass carp (Ctenopharyngodon idella), using a combination of PCR techniques: reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The resolved cDNA encompasses 2420 bp, containing an open reading frame corresponding to 324 amino acids. The deduced amino acid sequence shares high homology with those of mammalian desaturases. Northern blot and RT-PCR analyses demonstrated a high abundance of the transcript in liver tissue but low abundance in brain tissue. Furthermore, the structure of the gene has been resolved by screening its cognate genomic DNA library. The analysis shows that this gene is composed of six exons and five introns, encompassing a region of 8.5 kb. In particular, the last exon contains a length of the 3' untranslated region as long as 1382 bp. Although the primary sequence and the genomic organization are phylogenetically conserved between fish and mammals, the regulation of the gene expression appears to be divergent among species.