鲤食物转化率相关候选基因ffar3序列与表达分析

Sequence Identification and Expression of Food Conversion Efficiency Candidate Gene ffar3 in Common Carp Cyprinus carpio

FFAR3(Free fatty acid receptor 3)是一类短链脂肪酸和重要的能量来源,可调节机体内脂肪形成和动物肠道对营养物质的吸收。体质量约200 g的1龄鲤Cyprinus carpio投喂后0 h(对照组)、2 h、4 h和6 h分别取5尾鱼,用MS-222麻醉解剖取适量脑、肠、肝脏和肌肉等新鲜组织,用SuperScript Ⅲ First-Strand Synthesis System for RT-PCR试剂盒(Invitrogen)和定量PCR试剂盒One Step SYBR■Prime ScriptTM RT-PCR Kit Ⅱ(TaKaRa)等测定了鲤ffar3序列及其表达。结果表明:鲤ffar3基因全长为3318 bp,具有一个长度为990 bp的开放式阅读框,编码329个氨基酸;含有7个跨膜螺旋结构区域,与斑马鱼Danio rerio的相似性最高,达94.19%;与人类Homo sapiens、小鼠Mus musculus、半滑舌鳎Cynoglossus semilaevis、虹鳟Oncorhynchus mykis、斑点叉尾鮰Ictalurus punctatus、牙鲆Paralichthys olivaceus、大刺鳅Mastacembelus armatu及青鳉Oryzias melastigma等物种相似性差异不大,同源性介于70.37%~78.46%之间。分子进化树分析表明,所有硬骨鱼类ffar3基因与哺乳动物ffar3基因分属于系统树的2个分支。硬骨鱼类分支中,不同物种形成2个亚支。鲤ffar3基因聚在斑马鱼、剑尾鱼Xiphophorus couchianus、半滑舌鳎和斑点叉尾鮰的分支中。组织表达结果显示,ffar3基因在肠中表达量最高,肌肉和脑中次之,而在肝组织中表达量最小。进食后不同时间在脑、肠和肌肉中表达量差异显著,进食2 h脑中基因表达量最高,随着时间的推移,在4 h和6 h基因的表达量递减;而在肠和肌肉中,进食后2~6 h基因表达量升高,6 h时表达量最高。本研究结果表明:ffar3基因参与鲤摄食调控。

Free fatty acid receptor FFAR3 is known as a short-chain fatty acid, and an important energy source in animals and can regulates the body fat formation by regulation of the absorption of nutrients by animal intestines. The ffar3 sequence and its expression were analyzed in brain, intestine, liver and muscle of common carp(Cyprinus carpio)with body weight of about 200 g anesthetized by MS-222 at 0 h(control group), 2 h, 4 h and 6 h after the common carp fed using SuperScript Ⅲ First-Strand Synthesis System for RT-PCR kit(Invitrogen)and quantitative PCR kit One Step SYBR■ Prime ScriptTMRT-PCR Kit Ⅱ(TaKaRa). The results showed that ffar3 gene of common carp was 3 318 bp in full-length, with one open reading frame(ORF)spanning a region of 990 bp and encoding329 amino acids. The ffar3 had secondary structure dominated by 7 transmembrane helix regions, with the similarity with zebrafish(Danio rerio, 94.19%)in protein sequences;and shares 70.37% ~ 78.46% sequence identity with other species including human(Homo sapiens), mouse(Mus musculus), half smooth sole(Cynoglossus semilaevis), rainbow trout(Oncorhynchus mykis), channel catfish(Ictalurus punctatus), oliver flounder(Paralichthys olivaceus), loach(Mastacembelus armatu)and medaka(Oryzias melastigma).Phylogenetic tree analysis demonstrated that ffar3 genes of all teleost and mammalian belonged to two branches in the phylogenetic tree. In the branch of teleost, ffar3 gene of common carp was clustered into the branches of zebrafish, swordtail Xiphophorus couchianus, half smooth sole and channel catfish. Real-time PCR revealed that there was the maximal m RNA expression level of ffar3 in intestine, followed by muscle and brain and the minimal level in liver. No significant differences in m RNA expression of the gene were observed in brain, intestine and muscle tissues at different time after feeding, with the maximal gene expression level in brain 2 h after feeding, and then decrease 4 h and 6 h after feeding. In the intestine and muscle, however, the gene expression level was constantly increased from 2 h to 6 h after feeding, with the maximum 6 h after feeding. The findings indicate that ffar3 gene is involved in the regulation of feeding in common carp.