Cloning and characterization of ∆6/∆5 fatty acyl desaturase (Fad) gene promoter in the marine teleost Siganus canaliculatus

Abstract

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C PUFA precursors, and all genes encoding the key enzymes for LC-PUFA biosynthesis have been cloned and functionally characterized, which provides us a potential model to study the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. As the primary step to clarify such mechanisms, present research focused on promoter analysis of gene encoding ∆6/∆5 fatty acyl desaturase (Fad), a rate-limiting enzyme catalyzing the first step in the conversion of C PUFA to LC-PUFA. First, 2044 bp promoter sequence was cloned by genome walking, and the sequence from -456 bp to +51 bp was determined as core promoter by progressive deletion mutation. Moreover, binding sites of transcription factors (TF) such as CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), stimulatory protein 1 (Sp1), nuclear factor Y (NF-Y), activated protein 1 (AP1), sterol regulatory element (SRE), hepatocyte nuclear factor 4α (HNF4α) and peroxisome proliferator activated receptor γ (PPARγ) were identified in the core promoter by site-directed mutation and functional assays. Moreover, NF-1 and HNF4α were confirmed to interact with the core promoter region by gel shift assay and mass spectrometry. This is the first report of the promoter structure of a ∆6/∆5 Fad in a marine teleost, and a novel discovery of NF-1 and HNF4α binding to the ∆6/∆5 Fad promoter.

Pparγ Is Involved in the Transcriptional Regulation of Liver LC-PUFA Biosynthesis by Targeting the Δ6Δ5 Fatty Acyl Desaturase Gene in the Marine Teleost Siganus canaliculatus

“…A potential Pparγ binding site was predicted in the rabbitfish Δ6Δ5 fads2 promoter by using online software TRANSFAC® and JASPAR® ( Fig. 1) (Dong et al, 2018). To confirm the effects of Pparγ on the Δ6Δ5 fads2 promoter activity, the overexpression vector pcDNA3.1-pparγ was constructed by cloning the rabbitfish pparγ open reading frame (ORF) (GenBank: JF502072.1) DNA fragment into the pcDNA3.1 vector (Invitrogen).…”

“…The rabbitfish Δ6Δ5 fads2 promoter (2044 bp) was cloned from the Δ6Δ5 Fad mRNA of S. canaliculatus (GenBank: EF424276.2) (Dong et al, 2018). The promoter reporter vector was constructed with the Δ6Δ5 fads2 promoter fragment and pGL4.10, and the Δ6Δ5 fads2 Pparγ binding site-directed mutant of the Δ6Δ5 fads2 promoter was constructed with the mutation site in the middle of the primer.…”

“…Numbers are given relative to the first base of the transcription start site (TSS). Potential transcription binding motif for Pparγ is marked in grey (Dong et al, 2018). HEK 293T cells were co-transfected with the Δ6Δ5 fads2 promoter vector and the overexpression vector pcDNA3.1-pparγ or the empty vector pcDNA3.1 (control).…”

“…Particular progress on elucidating the mechanisms involved in fads2 regulation of teleosts has been made in rabbitfish S. canaliculatus, the first marine teleost demonstrated to have the ability of bioconverting C18 PUFA to LC-PUFA (Li et al, 2010, Li et al, 2008 and possessing two distinct Fads2 with Δ4 and Δ6Δ5 desaturase capabilities (Li et al, 2010). Thus, the expression of the rabbitfish fads2 is controlled at transcriptional level by Lxr and Srebp1, regulating both Δ4 and Δ6Δ5 fads2 in rabbitfish liver (Zhang et al, 2016a), and by hepatocyte nuclear factor alpha (HNF4α) (Dong et al, 2016), specifically targeting Δ4 fads2 expression. Moreover, unique post-transcriptional regulatory mechanisms have been further established, with miR-17 exhibiting its action directly on Δ4 fads2 (Zhang et al, 2014), and miR-33, whose more indirect action involves regulation of srebp1 (Zhang et al, 2016b).…”

Dietary Polyunsaturated Fatty Acid Supplementations Could Significantly Promote the Δ6 Fatty Acid Desaturase and Fatty Acid Elongase Gene Expression, Long Chain Polyunsaturated Fatty Acids, and Growth of Juvenile Cobia

“…Fish oil is rich in LC‐PUFA and the main source of LC‐PUFA of humans and other animals . However, the source of fish oil is limited with the rapid development of marine aquaculture . Fish oil has to be substituted for aquaculture and plant oil is a good substitute of fish oil .…”