中华鳖IGF2IGF2R基因克隆及其功能研究

Cloning and functional analysis of IGF2 and IGF2R genes in Chinese soft-shelled turtle(Pelodiscus sinensis

  • 摘要: 【目的】探索胰岛素样生长因子2基因(IGF2)和胰岛素样生长因子2受体基因(IGF2R)在中华鳖生长过程中发挥的作用,为揭示IGFs系统在中华鳖异速生长及两性生长差异中的作用机制打下基础。【方法】采用RACE克隆中华鳖IGF2IGF2R基因c DNA序列,利用BioEdit、ExPASy、TMHMM Server v2.0、SignalP及SMART等在线软件进行生物信息学分析,并通过实时荧光定量PCR分析IGF2IGF2R基因在雌雄中华鳖不同组织、不同表型及不同性类固醇激素处理下的表达变化。【结果】中华鳖IGF2基因cDNA序列全长1180 bp,共编码222个氨基酸残基;IGF2蛋白相对分子量为24.84 kD,理论等电点(pI)为9.00,属于跨膜蛋白,含有1个IIGF结构域。中华鳖IGF2R基因cDNA序列全长8765bp,共编码2443个氨基酸残基;IGF2R蛋白相对分子量为271.92kD,pI为5.64,属于跨膜蛋白,含有14个重复的CIMR结构域和1个FN2结构域。基于IGF2IGF2R氨基酸序列相似性构建的系统发育进化树均显示,中华鳖与龟鳖类的亲缘关系最近,与鱼类和两栖动物的亲缘关系相对较远。IGF2IGF2R基因在中华鳖体内具有广泛的组织表达谱,IGF2基因在精巢中的相对表达量显著高于卵巢(P<0.05,下同),IGF2R基因则恰好相反。IGF2基因在成年中华鳖生长快个体肝脏中的相对表达量显著高于生长慢个体,IGF2R基因的表达模式则相反。在雌二醇(E2)处理下,除雌性中华鳖肝脏中的IGF2R基因相对表达量在注射6 h时显著升高外,IGF2IGF2R基因在雌雄个体肝脏中的相对表达量整体上呈下降趋势;MT处理对雄性个体肝脏中IGF2基因的表达无显著影响(P>0.05),但在注射48 h后显著提高雌性个体肝脏中的IGF2基因相对表达量,且显著下调雌性中华鳖肝脏中的IGF2R基因相对表达量。【结论】IGF2IGF2R基因参与调控中华鳖性腺的发育及成熟,且IGF2R可能通过内化降解IGF2而对中华鳖的生长起负调控作用。此外,IGF2IGF2R基因会通过下调表达量来响应外源激素的刺激,进而实现对中华鳖生长的调控。

     

    Abstract: 【Objective】To explore the role of insulin-like growth factor 2 gene(IGF2) and insulin-like growth factor 2receptor gene(IGF2R) in the growth of Pelodiscus sinensis, and to lay a foundation for revealing the mechanism of IGFs system in the allometric growth and sexual growth difference of P. sinensis. 【Method】The cDNA sequences of IGF2 and IGF2R genes in P. sinensis were cloned by RACE. Bioinformatics analysis was performed using online softwares such as BioEdit, ExPASy, TMHMM Server v2.0, SignalP and SMART. The expression changes of IGF2 and IGF2R genes in different tissues, different phenotypes and different sex steroid hormones of male and female P. sinensis were analyzed by real-time fluorescence quantitative PCR. 【Result】The total length of the cDNA sequence of IGF2 gene was 1180 bp, encoding 222 amino acid residues. IGF2 protein, with a relative molecular weight of 24.84 kD and theoretical isoelectric point(pI) of 9.00, was a transmembrane protein and contained one IIGF domain. The total length of the cDNA sequence of IGF2R gene was 8765 bp, encoding 2443 amino acid residues. IGF2R protein had a relative molecular weight of 271.92 kD and a pI of 5.64. It was a transmembrane protein with fourteen repeated CIMR domains and one FN2 domain.Phylogenetic trees based on IGF2 and IGF2R amino acid sequence similarity showed that P. sinensis was most closely related to tortoises and far from fish and amphibians. IGF2 and IGF2R genes had a wide tissue expression profile in P. sinensis. The relative expression of the IGF2 gene in testis was significantly higher than that in the ovary(P<0.05, the same below), while the situation was the opposite for IGF2R gene. The relative expression of IGF2 gene in the liver of fast growing P. sinensis individuals was significantly higher than that in the slow-growing individuals, while the expression pattern of IGF2R gene was the opposite. Under estradiol(E2) treatment, except that the relative expression of IGF2R gene in the liver of female P. sinensis was significantly increased at 6 h after injection, the relative expression of IGF2 and IGF2R genes in the liver of male and female P. sinensis were decreased on the whole. MT treatment had no significant effect on IGF2 gene expression in male P. sinensis liver(P>0.05), but significantly increased the relative expression of IGF2 gene in female P. sinensis liver 48 h after injection, and significantly decreased the relative expression of IGF2R gene in female P. sinensis liver. 【Conclusion】IGF2 and IGF2R genes are involved in the regulation of the gonad development and maturation of P. sinensis, and IGF2R may negatively regulate the growth of P. sinensis by internalizing and degrading IGF2. In addition, IGF2 and IGF2R genes regulate the growth of P. sinensis by down-regulating their expression levels in response to exogenous hormone stimulation.

     

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