鳜论文鳜胰岛素样生长因子基因的克隆特征与表达分析.docVIP

鳜论文：鳜胰岛素样生长因子基因的克隆、特征与表达分析 【中文摘要】采用RT-PCR、cDNA末端快速扩增法(RACE)等分子克隆技术获得了鳜(Siniperca chuatsi)肝组织胰岛素样生长因子-Ⅰ和Ⅱ(Insulin-like growth factor-Ⅰ,Ⅱ)基因cDNA全长序列,并分析它们的结构特征;用实时荧光定量RT-PCR检测分析了它们在肝脏、肾、脑、前肠、后肠、性腺、胃、脾、鳃、心、肌肉组织,以及孵化后0、2、4、6、8、10、12、14、16、19天幼体中的表达特征。结果表明:(1)鳜胰岛素样生长因子-Ⅰ(IGF-Ⅰ)cDNA全长共1784bp,包括5’端非翻译区233bp,3’端非翻译区990bp和开放阅读框561bp,共编码186个氨基酸,由信号肽、成熟肽、E肽三部分组成,其中,信号肽44个氨基酸,成熟肽68个氨基酸,E肽74个氨基酸;成熟肽由B、C、A、D 4个区域组成,E肽分析表明,鳜IGF-Ⅰ属Ea-4型;鳜IGF-Ⅰ氨基酸序列与其他脊椎动物IGF-Ⅰ氨基酸序列相似度达81%～99%。鳜胰岛素样生长因子-Ⅱ(IGF-Ⅱ)cDNA全长共1643bp,包括5端非翻译区103bp,3’端非翻译区892bp和开放阅读框648bp,共编码215个氨基酸,由信号肽、成熟肽、E肽3部分组成,其中,信号肽47个氨基酸,成熟肽70个氨基酸,E肽98个氨基酸;鳜IGF-Ⅱ氨基酸序列与其它脊椎动物IGF-Ⅱ氨基酸序列相似度达到81%-98%,与鳜IGF-Ⅰ氨基酸序列相似度仅为62.5%。(2)IGF-ⅠmRNA在肝脏中表达量最高,肾、脑、后肠、性腺中表达量次之,胃、脾、前肠、鳃、心、肌肉中表达量较低;IGF-ⅡmRNA在在脑中表达量最高,肌肉、心、胃、鳃、肾中表达次之,脾、前肠、后场、性腺、肝中表达量较低。(3)幼体早期发育过程中,IGF-ⅠmRNA在孵化后0、2、4、6、8、10、12、14、16、19天各期幼体均有表达,表达量逐渐升高;IGF-ⅡmRNA在胚胎孵化后各期均有表达,孵化当天表达量最高,之后表达量略有降低。以上结果为进一步研究它们对鳜生长发育调控作用奠定了分子基础。 【英文摘要】In this study, the complete cDNA sequences of insulin-like growth factor-Ⅰand -Ⅱof Siniperca chuatsi liver were isolated by means of RT-PCR, rapid amplification of cDNA ends (RACE) and other cloning techniques, their structure characteristics were also analyzed. Using real-time quantitative RT-PCR, their expression level in different adult tissues and larval development stages (days after hatching of 0, 2, 4, 6, 8, 10, 12, 14, 16, 19) were also detected. Results showed that: (1) The complete cDNA of insulin-like growth factor-Ⅰwas 1784 bp, it contained a 233 bp 5′-untranslated region, 990 bp 3′-untranslated region and 561 bp open reading frame (ORF), which encoded 186 amino acids with a putative signal peptide of 44 amino acids, a mature peptides of 68 amino acids, and a E peptides of 74 amino acids, the mature peptides includes B, C, A and D domains, E peptides analysis showed that S. chuatsi IGF-Ⅰbelong to Ea-4 type. The similarity of S. chuatsi IGF-Ⅰamino acid sequence was 81%-99% with that of other v