草鱼胰岛素样生长因子1受体基因cDNA全序列的克隆及功能

cDNA cloning and function of insulin-like growth factor-I receptor gene(Igf-Ir) in grass carp(Ctenopharyngodon idellus)

本研究采用cDNA末端快速扩增法(RACE)克隆了草鱼(Ctenopharyngodon idellus)IGF-IR基因全长cDNA序列,并对该基因在草鱼不同时期胚胎和成鱼不同组织中的表达进行了分析。序列分析表明,草鱼IGF-IR基因cDNA序列全长5 741 bp,包括5′端非翻译区822 bp,3′端非翻译区581 bp,开放阅读框4 338 bp,共编码1 445个氨基酸。序列比对结果显示,草鱼IGF-IR可能属于a型,该基因编码的氨基酸序列与鲤(Cyprinus carpio)IGF-IRa、斑马鱼(Danio rerio)IGF-IRa和人类(Homo sapiens)IGF-IR的相似性分别为95%、93%和66%,具有较高的同源性,表明该基因在长期进化中具有较高的保守性。RT-PCR结果表明,该基因从16 hpf(hours post fertilization)胚胎期到出苗期都有表达,在成鱼大部分组织中均有表达。原位杂交结果显示,草鱼IGF-IR mRNA在不同时期胚胎组织中广泛存在,其中在脑部、脊索和尾部等生长旺盛组织的细胞中表达量较高。本研究为进一步探索草鱼IGF-IR基因在生长发育信号通路中的作用和育种提供了基础资料。

The insulin-like growth factor-I receptor （IGF-IR） is an important component of the insulin-like growth fac-tor system, which plays a key role in embryonic growth, formation of the nervous system, skeletal muscle development, and also appears to contribute to the establishment and progression of tumors. Despite its importance, little is known about the tissue distribution and expression pattern of IGF-IR during embryonic development in most commercially important cultured fish species. We isolated a 5 741 bp IGF-IR full-length cDNA from grass carp（Ctenopha ryngodon idellus）. The IGF-IR cDNA included an 822 bp complete 5′untranslated region （5′UTR）, a 581 bp 3′UTR, and a 4 338 bp open reading frame （ORF） region which encoded a 1 445 amino acid protein. The mature peptide contained seven structural domains including two Recep-L-domains, one Furin-like domain, three FN3 domains, and one PTKc-IGF-1R domain. Among these, the PTKc-IGF-1R domain was the most conservative domain of IGF-IR. The less conservative 3′UTR suggests that IGF-IR may play different transductional roles among different teleost species. The ORF deduced from the cDNA sequence was 95%, 93%, and 66%identical to the corresponding regions of IGF-IRa in common carp, zebrafish, and human IGF-IR, respectively, indicating a high level of conservation of IGF-IR during evolution. The grass carp IGF-IR has a high amino acid sequence identity with common carp and zebrafish IGF-IRa, suggesting it be-longs to the same type. Grass carp IGF-IR mRNA was detected during embryogenesis, beginning at 16 hpf （hours post fertilization） and continuing through the larval stages. The IGF-IR mRNA was transcribed in almost all adult tissues except the intestine, with expression being highest in the grass carp heart. Using whole mount in situ hybridization, IGF-IR mRNA was detected ubiquitously in 16 hpf embryos, with strong signals in the brain and spinal cord, but only a weak signal in the tail-fin. In 24 hpf embryos, the IGF-IR mRNA signal was strong in brain, spinal cord, and in rapidly growing regions such as tail-fin tip. Our results provide insight into the IGF-IR signaling pathways and provide a basis for future genetic breeding of grass carp.