基于4D-DIA蛋白质组学挖掘雌激素调控牛成肌细胞发育的关键蛋白  

Screening of key proteins in estrogen regulation of bovine myoblast development based on 4D-DIA proteomics

作　　者：宋霖节 孙圣杰 魏玉雯 海佳怡 喻宗岗 刘俊霞 任刚[1] SONG Linjie;SUN Shengjie;WEI Yuwen;HAI Jiayi;YU Zonggang;LIU Junxia;REN Gang(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;College of Life Sciences,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]西北农林科技大学生命科学学院,陕西杨凌712100

出　　处：《黑龙江畜牧兽医》2025年第3期1-12,共12页Heilongjiang Animal Science And veterinary Medicine

基　　金：陕西省人才资助项目(F2020123001);国家重点研发计划项目(2022YFF0001);秦创原人才项目(K3031222085)。

摘　　要：为了探究雌激素对牛成肌细胞发育的影响并挖掘相关蛋白与通路,试验首先通过在牛成肌细胞中加入不同浓度的17β-雌二醇(17β-estradiol,E2),研究其对牛成肌细胞发育的影响;然后利用4D-DIA蛋白质组学技术对E2处理(处理组)和未处理(对照组)的牛成肌细胞进行蛋白质丰度测定,筛选两组之间的差异表达蛋白(differentially expressed proteins,DEPs),并对其进行基因本体论(gene ontology,GO)功能注释与京都基因与基因组百科全书(Kyoto encyclopedia of genes and germoes,KEGG)富集分析;对DEPs中细胞组分进行亚细胞定位分析;通过蛋白质互作网络(protein protein interaction,PPI)分析挖掘DEPs之间的相互作用,鉴定核心蛋白;通过实时荧光定量PCR方法检测核心蛋白对应基因的mRNA表达水平,以验证蛋白质丰度测定结果。结果表明:10 nmol/L E2对牛成肌细胞发育具有一定的促进作用,共筛选到243个DEPs;与对照组比较,处理组有128个DEPs[CAMP依赖性蛋白激酶(PRKACA)、白细胞黏附分子(ALCAM)、丝裂原活化蛋白激酶12(MAP3K12)等]上调,115个DEPs[Rab家族11A(RAB11A)、β-1,3-半乳糖基转移酶相关蛋白(B3GNT9)、甾醇-27-羟化酶(CYP27A1)等]下调。这些DEPs显著富集在214个GO功能条目中,包括细胞过程、代谢过程、生物调节、生物过程调控等23个生物过程条目,细胞解剖实体和含蛋白复合物2个细胞组分条目,结合、催化活性、分子功能调节活性、转录调节因子活性等9个分子功能条目。DEPs富集到235条KEGG信号通路,根据显著性排名前15位的KEGG信号通路包括细胞黏附过程(甘露糖型O-聚糖的生物合成、细胞黏附分子)、细胞代谢过程(代谢途径、PPAR信号通路、色氨基酸代谢、精氨酸和脯氨酸代谢、氨基糖和核苷酸糖代谢、酪氨酸代谢)、细胞凋亡相关信号通路(P53信号通路)、机体系统(补体和凝血级联反应、胰腺分泌、初级胆汁酸生物合成、脂肪细胞In order to explore the effects of 17β-estradiol(E2)on the development of bovine myoblasts and to explore related proteins and pathways,in this experiment,firstly,the effect of estrogen on the development of bovine myoblasts was studied by adding different concentrations of estrogen to bovine myoblasts;then,4D-DIA proteomics technology was used to determine the protein abundance of E2-treated(treatment group)and untreated(control group)bovine myoblasts,and the differentially expressed proteins(DEPs)between the two groups were screened and GO function annotation and KEGG pathway enrichment analysis were performed.Subcellular localization analysis of cellular components in DEPs was carried out;protein-protein interaction(PPI)analysis was used to mine the interaction between DEPs for the identification of core proteins.The mRNA expression levels of the core protein-corresponding genes were detected by real-time quantitative PCR to verify determination results of the protein abundance.The results showed that 10 nmol/L E2 had a certain effect on the development of bovine myoblasts,and a total of 243 DEPs were screened.Compared with the control group,128 DEPs were up-regulated in the treatment group(CAMP-dependent protein kinase[PRKACA],leukocyte adhesion molecule[ALCAM],mitogen-activated protein kinase 12[MAP3K12],etc),and 115 DEPs were down-regulated(Rab family 11A[RAB11A],β-1,3-galactosyltransferase-related protein[B3GNT9],sterol-27-hydroxylase[CYP27A1],etc).These DEPs were significantly enriched in 214 GO functional items including 23 biological process items such as cellular process,metabolic process,biological regulation,biological process regulation,cell anatomical entity etc.,and 2 cell component items including cellular anatomical entity,protein-containing complex and 9 molecular function items,including binding,catalytic activity,molecular function regulation activity,and transcription regulator activity etc.DEPs were significantly enriched to 235 KEGG signaling pathways.The top 15 KEGG signaling pathways b

关 键 词：牛 成肌细胞 雌激素 蛋白组学 生物信息学 

分 类 号：S823.81[农业科学—畜牧学]