绵羊CREBRF基因克隆、生物信息学及组织表达分析

【目的】 对绵羊Luman/CREB3募集因子(CREBRF)基因进行克隆和生物信息学分析，并检测其在绵羊不同组织中的表达量，为探究CREBRF基因在绵羊中的生物学功能提供理论参考。【方法】 以绵羊卵巢cDNA为模板，通过PCR扩增和克隆绵羊CREBRF基因完整CDS区序列，并进行相似性比对、系统进化树构建及生物信息学分析;利用实时荧光定量PCR方法检测CREBRF基因在绵羊不同组织中的表达水平。【结果】 绵羊CREBRF基因CDS区序列全长1 920 bp，编码639个氨基酸。相似性比对结果表明，绵羊CREBRF氨基酸序列与山羊、牛、人、小鼠、猪、犬、马、鸡、鸭和斑马鱼的相似性分别为99.8%、99.1%、95.4%、93.6%、98.3%、97.5%、98.4%、88.0%、87.5%和61.3%。系统进化树分析结果显示，绵羊与山羊、牛的亲缘关系最近，与斑马鱼亲缘关系最远。生物信息学分析发现，绵羊CREBRF蛋白分子式为C₃₁₂₆ H₄₉₁₄ N₈₅₈ O₁₀₅₆ S₂₁，分子质量为72.08 ku，等电点(pI)为4.77，半衰期为30 h，肽链N-端为蛋氨酸(Met)，不稳定系数为54.83;CREBRF蛋白存在于细胞核内，不具备跨膜性，无信号肽，为亲水性不稳定蛋白。CREBRF蛋白二级结构主要以无规则卷曲(47.57%)为主，其次为α-螺旋(37.09%)。实时荧光定量PCR结果显示，CREBRF基因在绵羊不同组织中均有表达，其中在心脏、肾脏和卵巢中表达量显著高于其他组织(P<0.05)。【结论】 本试验获得了绵羊CREBRF基因CDS区全长序列，并初步研究了其组织表达规律，为研究绵羊胚胎发育的调控机制及提高繁殖力等提供了材料。

Cloning,Bioinformatics and Tissue Expression Analysis of CREBRF Gene in Ovis aries

【Objective】 The purpose of this study was to clone Luman/CREB3 recruitment factor(CREBRF) gene of Ovis aries and analyze its bioinformatics,and detect the expression of CREBRF gene in different tissues of Ovis aries,so as to provide theoretical reference for exploring the biological function of CREBRF gene in Ovis aries.【Methods】 Using ovary cDNA of Ovis aries as template,the complete CDS region of CREBRF gene was amplified by PCR and cloned,the similarity alignment,phylogenetic tree and bioinformatics analysis were carried out.The expression of CREBRF gene in different tissues of Ovis aries was detected by Real-time quantitative PCR.【Result】 The total length of CDS region of CREBRF gene was 1 920 bp,encoding 639 amino acids.The similarity alignment results showed that the similarity of CREBRF amino acid sequence of Ovis aries with Capra hircus,Bos taurus,Homo sapiens,Mus musculus,Sus scrofas,Canis lupus familiaris,Equus przewalskii,Gallus gallus,Anas platyrhynchos and Danio rerio were 99.8%,99.1%,95.4%,93.6%,98.3%,97.5%,98.4%,88.0%,87.5% and 61.3%,respectively.The phylogenetic tree analysis showed that Ovis aries had the closest relationship with Capra hircus and Bos taurus,and the furthest relationship with Danio rerio.Bioinformatics analysis found that the molecular formula of CREBRF protein in Ovis aries was C₃₁₂₆H₄₉₁₄N₈₅₈O₁₀₅₆S₂₁,the theoretical molecular weight was 72.08 ku,the theoretical isoelectric point (pI) was 4.77,the half-life was 30 h,the N-terminal of peptide chain was methionine (Met),and the instability coefficient was 54.83.CREBRF protein existed in the nucleus,there was no transmembrane property and signal peptide,and was a hydrophilic unstable protein.The secondary structure of CREBRF protein was mainly random random coil (47.57%),followed by alpha helix(37.09%).Real-time quantitative PCR results showed that CREBRF gene was expressed in different tissues of Ovis aries,the expression in heart,kidney and ovary were significantly higher than that in other tissues (P<0.05).【Conclusion】 This experiment obtained the full-length sequence of CREBRF gene,and preliminarily studied its tissue expression,which provided materials for studying the regulatory mechanism of embryonic development and improving fecundity in Ovis aries.