广西黄牛FGF2基因克隆、生物信息学分析及其在卵泡中的表达研究

doi:10.16431/j.cnki.1671-7236.2025.11.024

摘要/Abstract

摘要： 【目的】克隆广西黄牛成纤维生长因子2(fibroblast growth factor 2，FGF2)基因CDS区序列并进行生物信息学分析，探究FGF2基因在广西黄牛不同组织及卵泡中的表达规律，为进一步探讨FGF2基因在卵泡发育过程中的功能奠定基础。【方法】以广西黄牛卵巢组织cDNA为模板，克隆FGF2基因片段，与其他物种进行相似性比对及系统进化树构建，并通过在线软件对FGF2蛋白进行生物信息学分析。利用实时荧光定量PCR检测FGF2基因在广西黄牛不同组织中的表达水平，采用ELISA方法检测小(卵泡直径3～4 mm)、中(5～8 mm)、大(＞8 mm)卵泡卵泡液中的FGF2浓度，并结合免疫荧光技术及实时荧光定量PCR探究FGF2基因在广西黄牛卵泡中的定位及表达规律。【结果】广西黄牛FGF2基因CDS区全长468 bp，共编码155个氨基酸。广西黄牛FGF2蛋白氨基酸序列与猪、山羊、绵羊、兔的相似性超过98%，在哺乳动物中表现出高度保守性。生物信息学分析发现，广西黄牛FGF2蛋白为含14个磷酸化位点的碱性亲水蛋白，二级结构以无规则卷曲(61.94%)和延伸链(29.03%)为主，主要互作蛋白包括FGFR、PDGFRA、GPC1、EGFR等。组织表达分析发现，FGF2基因在广西黄牛各组织中均有表达，其中在卵巢和肺脏组织中的表达量显著高于其他组织(P＜0.05)。检测不同直径卵泡卵泡液中的FGF2浓度发现，直径＞8 mm卵泡卵泡液中的FGF2浓度显著高于3～4 mm卵泡(P＜0.05)。FGF2基因在卵母细胞中表达量显著高于颗粒细胞和卵泡膜细胞(P＜0.05)，其受体FGFR1基因在卵母细胞中表达量显著低于颗粒细胞和卵泡膜细胞(P＜0.05)。【结论】广西黄牛FGF2基因CDS区序列长468 bp，编码155个氨基酸，为稳定的碱性亲水蛋白，二级结构以无规则卷曲为主。FGF2基因在卵巢和肺脏组织中的表达量较高，在卵泡中主要来自卵母细胞，通过卵泡直径依赖性浓度变化调控卵泡发育。以上结果可为阐明FGF2基因在广西黄牛卵巢中的功能网络提供理论依据。

关键词: 广西黄牛; FGF2基因; 克隆; 生物信息学; 表达模式; 卵泡发育

Abstract: 【Objective】 This study aimed to clone the coding sequence (CDS) of the fibroblast growth factor 2 (FGF2) gene in Guangxi cattle and conduct bioinformatics analysis,investigate the expression patterns of FGF2 gene in various tissues and ovarian follicles,and establish a foundation for further exploration of its functional roles in follicular development. 【Method】 The FGF2 gene fragment was amplified from Guangxi cattle ovarian cDNA,followed by sequence alignment and phylogenetic tree construction with other species.Bioinformatics analysis of FGF2 protein was performed using online tools.The expression of FGF2 gene in different tissues of Guangxi cattle were detected via Real-time quantitative PCR.ELISA was employed to quantify FGF2 concentrations in follicular fluid from small (3-4 mm diameter),medium (5-8 mm),and large (＞8 mm) follicles.Immunofluorescence and Real-time quantitative PCR were combined to analyze the spatial localization and expression dynamics of FGF2 gene in follicular cells. 【Result】 The CDS of FGF2 gene in Guangxi cattle spanned 468 bp,encoding 155 amino acids.The amino acid sequence similarity of FGF2 protein in Guangxi cattle exhibited ＞98% with Sus scrofa,Capra hircus,Ovis aries,and Oryctolagus cuniculus,demonstrating high conservation among mammals.Bioinformatics analysis results revealed that FGF2 was an alkaline hydrophilic protein containing 14 phosphorylation sites,with secondary structures dominated by random coil (61.94%) and extended chain (29.03%),and key interacting partners included FGFR,PDGFRA,GPC1,and EGFR.Tissue expression analysis revealed that FGF2 gene was expressed in various tissues of Guangxi cattle,with significantly higher expression in ovarian and lung tissues compared with other tissues (P＜0.05).Follicular fluid FGF2 concentrations in large follicles (＞8 mm) were significantly elevated relative to small follicles (3-4 mm) (P＜0.05).The expression of FGF2 gene in oocytes was significantly higher than that in granulosa and theca cells (P＜0.05),while its receptor FGFR1 gene expression in oocytes was significantly lower than that in granulosa and theca cells (P＜0.05). 【Conclusion】 The CDS region of FGF2 gene in Guangxi cattle was 468 bp,encoding a polypeptide of 155 amino acids characterized as a stable alkaline hydrophilic protein,with its secondary structure dominated by random coil.FGF2 gene exhibited significantly higher expression in ovarian and lung.Within ovarian follicles,its expression was predominantly localized to oocytes,and it regulated follicular development via follicle diameter-dependent concentration dynamics.The results provided a theoretical foundation for elucidating the functional regulatory network of FGF2 gene in ovaries of Guangxi cattle.

Key words: Guangxi cattle; FGF 2 gene; cloning; bioinformatics; expression pattern; follicle development

中图分类号:

王云, 郑彦梓, 唐振华, 王燕新, 贾茹茹, 韦春烨, 周东平, 卢瑛, 黄荣春, 石德顺, 陆凤花. 广西黄牛FGF2基因克隆、生物信息学分析及其在卵泡中的表达研究[J]. 中国畜牧兽医, 2025, 52(11): 5276-5286.

WANG Yun, ZHENG Yanzi, TANG Zhenhua, WANG Yanxin, JIA Ruru, WEI Chunye, ZHOU Dongping, LU Ying, HUANG Rongchun, SHI Deshun, LU Fenghua. Cloning and Bioinformatics Analysis of FGF2 Gene in Guangxi Cattle and Its Expression Pattern in Ovarian Follicles[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5276-5286.

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