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 Δ9‐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. Mol. Reprod. Dev. 58:245–254, 2001. © 2001 Wiley‐Liss, Inc.

Isolation and molecular characterization of the porcine stearoyl-CoA desaturase gene

“…SCD-1 is mainly expressed in adipose tissues and liver (Ntambi et al, 1988), SCD-2 is principally expressed in brain (Kaestner et al, 1989), SCD-3 is skinspecifically expressed (Zheng et al, 2001), whereas SCD-4 is exclusively expressed in the heart . Similarly, SCD was only expressed in the liver and brain of the grass carp (Chang et al, 2001), and exclusively in the liver of the common carp (Polley et al, 2003). In contrast, SCD genes are ubiquitously expressed in human (Zhang et al, 1999), goat (Yahyaoui et al, 2002), sheep (Ward et al, 1998), zebrafish (Hsieh et al, 2003) and milkfish (Hsieh et al, 2001).…”

“…SCD genes have been so far characterized in human (Zhang et al, 1999), mouse (Ntambi et al, 1988;Kaestner et al, 1989), rat (Mihara, 1990), goat (Yahyaoui et al, 2002;Bernard et al, 2001), sheep (Ward et al, 1998), housefly (Eigenheer et al, 2002), zebrafish (Hsieh et al, 2003), milkfish (Hsieh et al, 2001), grass carp (Chang et al, 2001), and common carp (Polley et al, 2003). Prior to our project, a 1003-bp-long partial porcine SCD mRNA sequence was deposited in the database (GenBank accession no.…”

Molecular cloning and expression analysis of scd1 gene from large yellow croaker Larimichthys crocea under cold stress

“…SCD1 is mainly expressed in the liver, whereas SCD2 and SCD5 are mainly expressed in the brain, and SCD3 and SCD4 are tissue-specific proteins expressed in the skin and heart, respectively (Hodson and Fielding, 2013;Lengi and Corl, 2007;Miyazaki et al, 2003;Ntambi and Miyazaki, 2003;). In fish, only two SCD isoforms, SCD1 and SCD2 were found in grass carp (Ctenopharyngodon idella), and were highly expressed in the liver but low in the brain (Chang et al, 2001;Polley et al, 2003;Evans et al, 2008). In tilapia, SCD1 was only expressed in the liver (Hsieh et al, 2004), while in milkfish and zebrafish, SCD1 was found in various tissues (Hsieh et al, , 2003.…”

Identification and Functional Expression of a Δ9‐Fatty Acid Desaturase from Psychrobacter urativorans in Escherichia coli

“…In this sense, besides the soluble D9-ACP desaturases, the other two classes of membrane-bound D9-desaturases in different species could be regarded as alternative desaturase resource for more efficient production of PUFAs. However, although most research has been done on the D9 acyl-lipid desaturase isolated from cyanobacteria [31][32][33][34], the number of prokaryotic D9-desaturase enzymes that have been investigated in detail is much smaller than those from fungi [35,36], higher plants [10,37], fish [38] and mammals [39] so far. In the present study, a novel gene that encodes a membrane-bound D9-fatty acid desaturase from P. urativorans has been cloned and, to our knowledge, for the first time, successfully expressed in E. coli.…”