草鱼和翘嘴鲌fgfrhl-1基因的克隆和表达分析

成纤维细胞生长因子受体同源类似物1(fgfrhl-1)基因是目前仅在鱼类基因组中检测到的fgfr基因家族成员, 该序列在鱼类进化过程中高度保守。为研究fgfrhl-1基因的表达情况和具体的功能, 在亲缘关系较远的草鱼(Ctenopharyngodon idellus)和翘嘴鲌(Culter alburnus Basilewsky)中克隆了fgfrhl-1的cDNA序列, 并通过半定量RT-PCR和冰冻切片原位杂交分析了该基因在成体不同组织中的表达情况。克隆结果的序列分析表明: 草鱼fgfrhl-1 cDNA序列全长为1472 bp, 5′-UTR长213 bp, 3′-UTR长56 bp, 开放阅读框长1203 bp; 翘嘴鲌fgfrhl-1 cDNA序列全长为1886 bp, 5′-UTR长298 bp, 3′-UTR长385 bp, 开放阅读框长1203 bp。在两种鱼类中该基因都编码400个氨基酸, 其预测的氨基酸序列同源性高达95.5%。蛋白二级结构预测表明Fgfrhl-1具有FGFRs家族蛋白的胞内酪氨酸激酶区, 跨膜的螺旋区和胞外配体识别结合区, 但其胞外区比FGFRs缺少了3个免疫球蛋白样结构域。通过RT-PCR方法在两种鱼类的心脏、鳃、肝、脾、尾鳍以及肌肉组织的肌间隔中均检测到了fgfrhl-1表达, 但在肌纤维中均没有检测到其表达。对这两种鱼类的肌肉组织、肝脏和脾脏进行的组织切片原位杂交表明fgfrhl-1只在这些组织和器官的结缔组织及导管中表达, 不在间质细胞结构中表达。这些结果说明: fgfrhl-1的成体组织特异性表达模式在不同鱼类中基本一致, fgfrhl-1在鱼类各组织和器官的结缔组织和导管的细胞中表达, 不在间质细胞中表达。因此, fgfrhl-1可能在鱼类结缔组织及导管分化调控或功能维持中有独特作用。

CLONE AND EXPRESSING ANALYSIS OF FGFRHL-1 IN CTENOPHARYNGODON IDELLUS AND CULTER ALBURNUS

Fibroblast growth factor receptor homolog 1 (fgfrhl-1) gene is a member of the fgfr gene family currently detected only in the genomes of fish. The sequence of fgfrhl-1 is highly conserved during fish evolution, however, the distribution of fgfrhl-1 and its specific function remains unclear. In this study, we cloned the full-length cDNA of fgfrhl-1 from two distant fish species, the grass carp, Ctenopharyngodon idellus, and the Culter alburnus, and analyzed its expression in various adult tissues. The results showed that the full-length of fgfrhl-1 cDNA sequence of grass carp was 1472 bp, containing a 213 bp 5′-UTR, a 1203 bp open reading frame and a 56 bp 3′-UTR. The full-length fgfrhl-1 cDNA sequence of C. alburnus was 1886 bp, containing a 298 bp 5′-UTR, a 1203 bp open reading frame and a 385 bp 3′-UTR. The gene identified in both fish species encoded 400 amino acids, and the homology of the predicted amino acids sequences between them was 95.5%. The secondary structure prediction revealed that Fgfrhl-1 in both fish species has an intracellular tyrosine kinase domain, a transmembrane helix domain and an extracellular ligand recognizing and binding domain similar to those of FGFR, and that the extracellular domain of Fgfrhl-1 lacks three Ig-like domains that included in classical FGFRs. Semi-quantitative RT-PCR analysis explored that fgfrhl-1 was expressed in heart, gill, liver, spleen, caudal fin and intermuscular septum but not in muscle fibers in both fish species. In situ hybridization results revealed that fgfrhl-1 was expressed in connective tissues and vessels but not in the structure formed by mesenchymal cells. These results indicated the similar expression pattern of fgfrhl-1 of two different fish species mainly in the connective tissue and vessel cells of various tissues and organs, indicating that fish fgfrhl-1 may play a unique role in regulating differentiation or function maintenance of connective tissues and vessels.