秦川肉牛PAI 1基因生物信息学分析及其对肌内脂肪细胞增殖和分化的影响  

Bioinformatics Analysis of PAI 1 Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Adipocytes in Qinchuan Beef Cattle

作　　者：张殿琦 马鑫浩 杨志梅 梅楚刚 昝林森[1,2] ZHANG Dianqi;MA Xinhao;YANG Zhimei;MEI Chugang;ZAN Linsen(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;National Beef Cattle Improvement Center,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]国家肉牛改良中心,杨凌712100

出　　处：《中国畜牧兽医》2023年第5期1729-1741,共13页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31972994);国家肉牛牦牛产业技术体系(CARS-37);中央引导地方科技发展专项(2060404-51301)。

摘　　要：【目的】对秦川肉牛纤溶酶原激活物抑制剂1(plasminogen activator inhibitor 1,PAI1)基因进行生物信息学分析,检测PAI 1基因在秦川肉牛不同组织中的表达水平并研究其功能,为秦川肉牛肌内脂肪沉积的分子育种提供理论依据。【方法】以分离自秦川肉牛背最长肌的肌内脂肪细胞为试验材料,PCR扩增PAI 1基因CDS区,对所获序列进行相似性分析及系统进化树构建,并通过生物信息学软件预测其编码蛋白的结构和功能。通过实时荧光定量PCR检测PAI 1基因在秦川肉牛不同组织中的表达量。合成PAI 1基因siRNA并转染秦川肉牛肌内脂肪细胞,试验分为两组:对照组(NC)和干扰组(si-PAI1),通过EdU染色、CCK-8检测、流式细胞术、油红O染色观察表型变化,采用实时荧光定量PCR及Western blotting检测转录和翻译水平标志基因(PCNA、CDK1、CCND2、PPARG、PLIN2、FABP4)的表达。【结果】试验成功扩增出PAI 1基因CDS区,大小为1054 bp。牛PAI 1基因氨基酸序列与水牛、山羊、人、小鼠、绵羊、黑猩猩的相似性分别为96.8%、95.8%、85.3%、76.9%、95.8%和85.3%。系统进化树表明,牛与水牛的亲缘关系最近,与小鼠的亲缘关系最远。生物信息学分析发现,细胞周期相关激酶参与了PAI 1基因的磷酸化修饰;PAI1蛋白二级结构主要以α-螺旋和无规则卷曲为主;PAI 1基因启动子上游2000 bp存在成脂相关转录因子的结合位点。实时荧光定量PCR结果发现,PAI 1基因在秦川肉牛各组织中均有表达,其中在肾周脂肪中的表达量最高,在背最长肌中的表达量最低。干扰PAI 1基因后,与对照组相比,干扰组处于增殖期的细胞数量明显增多,CDK 1、PCNA和CCND 2基因mRNA表达水平极显著或显著上调(P<0.01;P<0.05),CDK1和PCNA蛋白表达水平明显增加。随着肌内脂肪细胞的分化,与对照组相比,干扰组脂滴数量明显增加,PPARγ、PLIN 2和FABP 4基因mRNA表达水平均极显著上调(P<0.01【Objective】The purpose of this experiment was to conduct bioinformatics analysis of plasminogen activator inhibitor 1(PAI1)gene in Qinchuan beef cattle,detect its expression in different tissues and confirm its function by experiments,which provided the theoretical basis for molecular breeding to promote intramuscular fat deposition in Qinchuan beef cattle.【Method】Intramuscular adipocytes were isolated from longissimus dorsi muscle of Qinchuan beef cattle as the research object.The CDS region of PAI 1 gene was amplified by PCR.The similarity was analyzed and the phylogenetic tree was constructed,the structure and function of the encoding protein of PAI 1 gene was predicted by bioinformatics software.The expression of PAI 1 gene in different tissues of Qinchuan beef cattle was detected by Real-time quantitative PCR.The intramuscular adipocytes was transfected with siRNA of PAI 1 gene,the experiment was divided into two groups:Control group(NC)and interference group(si-PAI1).Flow cytometry,EdU staining,CCK-8 assay,and Oil red O staining were used to detect the cell phenotypic alterations.Real-time quantitative PCR and Western blotting were used to identify the expression of marker genes(PCNA,CDK1,CCND2,PPARG,PLIN2 and FABP4)at the transcriptional and translational levels.【Result】The CDS region(1054 bp)of PAI 1 gene was successfully amplified.The similarity of amino acid sequence of PAI1 protein between Bos taurus and Bubalus bubalis,Capra hircus,Homo sapiens,Mus musculus,Ovis aries and Pan troglodytes were 96.8%,95.8%,85.3%,76.9%,95.8%and 85.3%,respectively.The phylogenetic tree results showed that Bos taurus had the closest genetic relationship with Bubalus bubalis,and the farthest genetic relationship with Mus musculus.Bioinformatics prediction showed that cell cycle-related kinase was involved in the phosphorylation of PAI 1 gene.The secondary structure of PAI1 protein were mainly alpha helix and random coil.The binding sites of adipogenic transcription factors were also identified in the upstream 20

关 键 词：秦川肉牛 PAI 1基因 生物信息学 肌内脂肪细胞 

分 类 号：Q78[生物学—分子生物学]