翘嘴鲌胰岛素样生长因子igf3基因的克隆及表达分析

CLONE AND EXPRESSION ANALYSIS OF INSULIN-LIKE GROWTH FACTOR 3(IGF3)GENE IN CULTER ALBURNUS

为研究翘嘴鲌(Culter alburnus Basilewsky)胰岛素样生长因子(Insulin-like growth factor 3,igf3)基因在性别调控过程中的作用,基于分子克隆手段RT-PCR和RACE技术相结合方法扩增到翘嘴鲌igf3基因完整cDNA序列,利用qRT-PCR技术检测其在不同组织中的表达水平,最后通过甲基化检测方法比较分析了翘嘴鲌性腺组织igf3基因组水平的CpG岛修饰水平。实验结果显示igf3 cDNA序列全长901 bp,包含92 bp的5′端非编码区和203 bp的3′端非编码区,开放阅读框606 bp,编码201个氨基酸。序列分析显示,类似于其他IGF家族成员igf1和igf2,igf3同样存在保守的特征结构域,主要划分为前体信号肽、B、C、A、D和E区。igf3基因在翘嘴鲌不同组织中的表达水平差异明显(P<0.05),在卵巢中表达量最高,其次是精巢组织,而在脑、心脏、脾脏、肝脏、肌肉和肾脏中表达丰度极低。甲基化检测结果显示在卵巢中CG位点几乎不发生甲基化,然而精巢中的甲基化程度非常高,这与其表达水平正好呈负相关。研究结果表明igf3可能参与了性腺的形成或功能维持,igf3基因组启动子区域的DNA甲基化修饰与其在性腺组织的二态性表达特征关系密切。

Insulin-like growth factors(Igf)are evolutionarily conserved gene family that regulate a variety of cellular biological processes,including growth,proliferation,survival,migration,and differentiation.To understand the role of igf3 gene in sex determination and differentiation in Culter alburnus,the full-length cDNA sequence of igf3 was cloned by rapid amplification of cDNA ends(RACE)technology;real-time quantitative PCR(qRT-PCR)was employed to analyze the expression level of igf3 in different tissues;DNA sequencing of sodium bisulfite was used to explore the CpG methylation pattern of igf3 promoter in adult testes and ovaries.The length of igf3 cDNA was 901 bp with a 92 bp 5′UTR,a 203 bp 3′UTR and a 606 bp ORF encoding of 201 amino acid residues.Sequence analysis showed that similar to other IGF family members igf1 and igf2,igf3 also had conserved characteristic domains,which were mainly divided into precursor signal peptides,B,C,A,D,and E regions.The expression level of igf3 gene was significantly different in various tissues(P<0.05).The expression was the highest in the ovary,followed by the testis,while the expression abundance was extremely low in the brain,heart,spleen,liver,muscle and kidney.Analysis of CpG islands methylation pattern revealed that igf3 promoter CpGs were not methylated in the ovary,but is hypermethylated in the testis.These findings indicated that igf3 was involved in gonadal formation or functional maintenance in C.alburnus,and the DNA methylation modification in the promoter region of igf3 genome was closely related to sexual dimorphism expression between gonadal tissues.