关中奶山羊FGF2基因克隆、生物信息学分析及组织表达研究

【目的】对关中奶山羊成纤维细胞生长因子2（fibroblast growth factor 2,FGF2）基因进行克隆及生物信息学分析,并检测其在山羊各组织中的表达差异,为后续探究该基因的功能奠定基础。【方法】以关中奶山羊乳腺cDNA为模板,采用RT-PCR技术扩增并克隆关中奶山羊FGF2基因完整CDS区序列,进行相似性比对、系统发育树构建及生物信息学分析;运用实时荧光定量PCR方法检测FGF2基因在关中奶山羊心脏、肝脏、脾脏、肾脏、背最长肌、肺脏、乳腺和卵巢组织中的表达情况。【结果】关中奶山羊FGF2基因编码区长468 bp,可编码155个氨基酸,分子质量为17.26 ku,理论等电点为9.58,其中含量最高的是甘氨酸,占10.3%。相似性比对结果表明,关中奶山羊FGF2氨基酸序列与人、牛、马、兔、绵羊、虎鲸和野猪的相似性分别为98.7%、99.4%、100.0%、98.7%、99.4%、99.4%和62.6%。系统进化树显示,FGF2基因在不同物种间具有高度保守性,与马的亲缘关系最近。关中奶山羊FGF2蛋白不稳定指数为38.39,属于稳定亲水性蛋白质,不含跨膜结构和信号肽;FGF2蛋白二级结构含有α-螺旋、β-转角、延伸链、无规则卷曲,分别占比10.32%、14.19%、30.97%、44.52%。蛋白质互作预测分析显示,FGF2蛋白与FGFR、KDR、FGF1、FGFR4、MET和FGFR1等与乳腺生长发育相关的蛋白存在相互作用。实时荧光定量PCR检测到关中奶山羊FGF2基因在心脏、肝脏、脾脏、肾脏、背最长肌、肺脏、乳腺和卵巢中均有表达,在乳腺中表达水平最高,其次为卵巢,在背最长肌中的表达水平最低。【结论】关中奶山羊FGF2基因CDS区序列长468 bp,编码155个氨基酸,为亲水蛋白,二级结构以延伸链和无规则卷曲为主。FGF2基因在关中奶山羊泌乳高峰期的不同组织中均有表达。本试验结果为进一步探究FGF2基因在关中奶山羊乳腺发育中的作用及其具体功能提供了参考。

Cloning,Bioinformatics Analysis and Tissue Expression Study of FGF2 Gene in Guanzhong Dairy Goats

【Objective】 The purpose of this study was to perform cloning and bioinformatic analysis of fibroblast growth factor 2 (FGF2) gene in Guanzhong dairy goats, and detect the expression differences of FGF2 gene in various tissues of goats, so as to lay the foundation for the subsequent exploration of the function of FGF2 gene.【Method】 Using mammary gland cDNA of Guanzhong dairy goat as template, the complete CDS region of FGF2 gene was amplified by RT-PCR and cloned, the similarity alignment, phylogenetic tree and bioinformatics analysis were carried out.Meanwhile, Real-time quantitative PCR was used to detect the expression of FGF2 gene in heart, liver, spleen, kidney, longissimus dorsi muscle, lung, mammary gland and ovary of Guanzhong dairy goats.【Result】 The coding region of FGF2 gene in Guanzhong dairy goats was 468 bp, encoding 155 amino acids, the molecular mass was 17.26 ku and the theoretical isoelectric point was 9.58, the highest content was glycine, accounting for 10.3%.The similarity alignment results showed that the similarity of FGF2 amino acid sequence of Guanzhong dairy goat with Homo sapiens, Bos taurus, Equus caballus, Oryctolagus cuniculus, Ovis aries, Orcinus and Sus scrofa were 98.7%, 99.4%, 100.0%, 98.7%, 99.4%, 99.4% and 62.6%, respectively.The phylogenetic tree showed that FGF2 gene was highly conserved among different species and had the closest homologous relationship with Equus caballus.The instability index of FGF2 protein of Guanzhong dairy goat was 38.39, which was an stable hydrophilic protein without signal peptide and transmembrane domain.The secondary structure of FGF2 protein contained alpha helix, beta turn, extended chain and random coil, accounting for 10.32%, 14.19%, 30.97% and 44.52%, respectively.Protein interaction prediction analysis found that FGF2 protein interacted with FGFR, KDR, FGF1, FGFR4, MET and FGFR1 proteins related to mammary gland growth and development.Real-time quantitative PCR detected that FGF2 gene of Guanzhong dairy goat was expressed in heart, liver, spleen, kidney, longissimus dorsi muscle, lung, mammary gland and ovary.The expression in mammary gland was highest, followed by ovary, and the lowest expression in longissimus dorsi muscle.【Conclusion】 The CDS region of FGF2 gene sequence in Guanzhong dairy goat was 468 bp, and encoded 155 amino acids, which was a hydrophilic protein.The secondary structure of FGF2 protein was mainly extended chain and random coil.FGF2 gene in Guanzhong dairy goat was expressed in different tissues during the peak lactation period.The results of this study provided a reference for further exploring the role of FGF2 gene in Guanzhong dairy goat mammary gland development and its specific functions.