鲇POMC cDNA的克隆与表达分析

Molecular cloning of propiomelanocortin cDNA and multi-tissue mRNA expression in Parasilurus asotus

采用RT-PCR和cDNA末端快速扩增法(RACE)分离、克隆出鲇(Parasilurus asotus)垂体阿黑皮素原(proopiomelanocortin,POMC)基因全长cDNA序列,共1 099 bp,包括129 bp 5′非翻译区、648 bp阅读框以及322 bp 3′非翻译区,阅读框共编码215个氨基酸,POMC前体蛋白的结构主要由信号肽(Met1-Ala28)、N端多肽(Gln29-Asp102)、促肾上腺皮质激素(ACTH,Ser 105-Met144)、α-黑色素细胞刺激激素(α-MSH,Ser105-Val 117)、促肾上腺皮质激素样中叶肽(CLIP,Arg123-Met144)、γ-促脂解激素(γ-LPH,Glu145-Ser158)、β-黑色素细胞刺激激素(β-MSH,Asp161-Ser177)和β-内啡肽(β-EP,Tyr178-Leu215)构成。分子量计算值为24.66 kD,理论等电点为7.25。同源性分析表明鲇与斑点叉尾的相似性高达88%,其构成多肽中α-MSH、β-MSH、β-EP、CLIP保守性逐渐降低,而γ-MSH、连接肽(JP)、γ-LPH是变异比较大的区域,同时鲇POMC中不含有γ-MSH,连接肽和部分γ-LPH也不存在。生物信息学分析显示鲇POMC基因所编码的蛋白质以亲水性区域为主,具有丰富的B细胞抗原位点。定量分析结果表明POMC mRNA主要在垂体中高表达,在非垂体组织的脑中表达量最高,鳃、头肾、肝、脾、肌肉、皮肤等组织也有少量表达。

We investigated the structure,evolution,and tissue specific expression of Parasilurus asotus proopiomelanocortin（POMC） cDNA using RT-PCR and RACE.The POMC cDNA was 1 099 bp in length and contained a 129 bp 5′UTR,a 322 bp 3′UTR,and a 648 bp open reading frame encoding a protein of 215 amino acids.The catfish POMC protein contained a signal peptide（SP,Met1-Ala28）,an N-terminal peptide（Gln29-Asp102）,adrenocorticotropic hormone（ACTH,Ser105-Met144）,α-melanocyte stimulating hormone（α-MSH,Ser105-Val117）,corticotropin-like intermediate lobe peptide（CLIP,Arg123–Met144）,β-lipotropin（β-LPH,Glu145-Ser158）,β-MSH（Asp161-Ser177）,and β-Endorphin（β-EP,Tyr178-Leu215）.Catfish POMC protein does not contain a γ-MSH region and the majority of the joining peptide and part of the γ-LPH were deleted.The protein shared the highest similarity（88%） with channel catfish（Parasilurus asotus） POMC.The most highly conserved active peptide was α-MSH,followed by β-MSH,β-EP,and CLIP whereas γ-MSH,joining peptide（JP）,and γ-LPH were located in the most divergent region.The protein had a calculated molecular weight of 24.66 kD,a theoreti-cal isoelectric point of 7.25,was hydrophilic in most regions,and was rich in B cell antigenicity positions.Catfish POMC mRNA was primarily expressed in the pituitary and the concentration of mRNA was much higher in brain than in other tissues.