Dong Yewei, Zhao Jianhong, Chen Junliang, Wang Shuqi, Liu Yang, Zhang Qinghao, You Cuihong, Monroig Óscar, Tocher Douglas R, Li Yuanyou
School of Marine Sciences, South China Agricultural University, Guangzhou, Guangdong, China.
Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, Guangdong, China.
Gene. 2018 Mar 20;647:174-180. doi: 10.1016/j.gene.2018.01.031. Epub 2018 Jan 9.
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.
黄斑蓝子鱼是首个被证明具有从C多不饱和脂肪酸前体生物合成长链多不饱和脂肪酸（LC-PUFA）能力的海洋硬骨鱼，并且所有编码LC-PUFA生物合成关键酶的基因都已被克隆并进行了功能表征，这为我们提供了一个研究硬骨鱼中LC-PUFA生物合成调控机制的潜在模型。作为阐明此类机制的第一步，目前的研究集中在对编码∆6/∆5脂肪酸去饱和酶（Fad）基因的启动子分析上，该酶是催化C多不饱和脂肪酸转化为LC-PUFA第一步的限速酶。首先，通过基因组步移克隆了2044 bp的启动子序列，并通过逐步缺失突变将-456 bp至+51 bp的序列确定为核心启动子。此外，通过定点突变和功能分析在核心启动子中鉴定了转录因子（TF）如CCAAT增强子结合蛋白（C/EBP）、核因子1（NF-1）、刺激蛋白1（Sp1）、核因子Y（NF-Y）、活化蛋白1（AP1）、固醇调节元件（SRE）、肝细胞核因子4α（HNF4α）和过氧化物酶体增殖物激活受体γ（PPARγ）的结合位点。此外，通过凝胶迁移试验和质谱法证实NF-1和HNF4α与核心启动子区域相互作用。这是关于海洋硬骨鱼中∆6/∆5 Fad启动子结构的首次报道，也是NF-1和HNF4α与∆6/∆5 Fad启动子结合的新发现。
