| 摘要: |
| 胰岛素样生长因子-I（Insulin-like growth factor-I，IGF-I）是影响脊椎动物生长、发育及代谢的重要调控因子。本研究采用RT-PCR和RACE技术，克隆了银鲳（Pampus argenteus）肝脏IGF-IcDNA序列，应用半定量RT-PCR、Real-time qPCR和原位杂交的方法检测了IGF-I的组织表达特性、在肝脏中的生长表达特性和IGF-I基因在肝脏中的定位。序列分析表明，IGF-I cDNA序列全长836bp，其5'非编码区128bp、3'非编码区92bp，开放阅读框（open reading frame，ORF）605bp，由此推导IGF-I前体蛋白由201个氨基酸组成；前体肽由信号肽、成熟肽、E肽三部分组成，其中信号肽59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四个区域组成，E肽分析表明，银鲳IGF-I属Ea-4型。同源性比较结果表明，银鲳与同目鲈形目鱼类的IGF-I编码序列同源性较高，为83.52%-91.40%；与哺乳类、鸟类和爬行类的同源性较低。半定量RT-PCR和Real-time qPCR组织特异性表达结果显示，IGF-I mRNA在肝脏组织中的表达量最高，显著高于其它组织，肾脏、心脏、肌肉、脑、鳃、小肠、卵巢次之，嗅球、脾脏和胃中表达较低；半定量RT-PCR和Real-time qPCR不同生长阶段表达结果显示，IGF-I mRNA在30-50g肝脏组织中表达量最高（P<0.05）；IGF-I mRNA在肝脏中的原位杂交定位结果显示，在肝脏细胞中均有表达，阳性信号主要位于细胞质中，靠近细胞边缘处信号较强。 |
| 关键词: 银鲳 胰岛素样生长因子-I基因 荧光定量 原位杂交 |
| DOI：10.11693/hyhz20160600142 |
| 分类号: |
| 基金项目:国家星火计划项目，2015GA701007号；浙江省自然科学基金项目，LY15C190003号；宁波市重大科技专项，2015C110003号。 |
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| CLONG AND EXPRESSION ANAYSIS OF INSULIN-LIKE GROWTH FACTOR I GENE IN PAMPUS ARGENTEUS |
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ZHU Xiao-Fang, GUO Xiao-Ge, ZHANG Ding-Yuan, YANG Jia-Zhe, XU Shan-Liang, WANG Dan-Li
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School of Marine Science, Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China
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| Abstract: |
| Insulin-like growth factor-I (IGF-I) is an important regulator to growth,development,and metabolism of a vertebrate.To clone the IGF-I cDNA sequences in liver of Pampus argenteus,we used RT-PCR (reverse transcription-polymerase chain reaction),RACE (rapid amplification of cDNA ends).In addition,to detect the IGF-I expression in the tissues and IGF-I expression levels in the liver in six different growth stages and the positioning of the IGF-I gene in the liver,we used semi-quantitative RT-PCR,quantitative RT-PCR,and in-situ hybridization.Results show that the IGF-I cDNA precursor consists of 836bp with a 605bp open reading frame,ORF,and 5'and 3'UTR (untranslated regions,128bp and 92bp,respectively).Therefore,we deduce that the precursor protein of IGF-I consisted of 201 amino acids;the precursor peptide is composed of signal peptide,mature peptide and E peptide,signal peptide of 59 amino acids,mature peptide of 68 amino acids,and E peptide of 74 amino acids.The mature peptide was composed of four regions B,C,A,and D.Analysis on E peptide showed that IGF-I of pomfret was in Ea-4 type.The IGF-I coding sequence of pomfret had higher identity with the same mesh Perciformes fish,from 83.52% to 91.40%;and lower identity with mammals,birds and reptiles.Semi-quantitative RT-PCR and RT qPCR results show that IGF-I mRNA was expressed in all 10 tissues,high in liver,and low in other tissues.Semi-quantitative RT-PCR and quantitative RT PCR in six different growth stages showed that IGF-I mRNA expression level was the highest when fish weighed between 30-50g,and then decreased significantly between 50-100g (P<0.05).The in-situ hybridization to see IGF-I mRNA expression in the liver indicated that GF-I positive cells were found throughout the liver;the signal intensity was significantly higher at cell edge,and positive signals were found mainly in cytoplasm. |
| Key words: Pampus argenteus insulin-like growth factor-I gene quantitative real-time PCR in situ hybridization |
