秦川肉牛PAI1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

doi:10.16431/j.cnki.1671-7236.2023.05.001

Abstract

Abstract: 【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 PAI1 gene was amplified by PCR.The similarity was analyzed and the phylogenetic tree was constructed,the structure and function of the encoding protein of PAI1 gene was predicted by bioinformatics software.The expression of PAI1 gene in different tissues of Qinchuan beef cattle was detected by Real-time quantitative PCR.The intramuscular adipocytes was transfected with siRNA of PAI1 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 (1 054 bp) of PAI1 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 PAI1 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 2 000 bp of PAI1 gene promoter.Real-time quantitative PCR results showed that PAI1 gene was expressed in all tissues,with the highest expression in perirenal fat and the lowest expression in longissimus dorsi muscle.After interfering with PAI1 gene,compared with control group,the cell number of the proliferative phase in interference group was significantly increased,the mRNA expression of CDK1,PCNA and CCND2 genes were extremely significantly or significantly increased (P＜0.01 or P＜0.05),and the protein expression of CDK1 and PCNA were also significantly increased.With the differentiation of intramuscular adipocytes,compared with control group,the number of lipid droplets in interference group was significantly increased,and the mRNA expression of PPARγ,PLIN2 and FABP4 genes were extremely significantly increased (P＜0.01).【Conclusion】 PAI1 gene was highly conserved among species.PAI1 might participate in the proliferation and differentiation of intramuscular adipocytes through phosphorylation and transcription factors.The interfering with PAI1 gene could promote the proliferation and differentiation of intramuscular adipocytes,which suggested that PAI1 gene might play an important role in regulating the deposition of intramuscular fat.

Key words: Qinchuan beef cattle; PAI1 gene; bioinformatics; intramuscular adipocytes

CLC Number:

ZHANG Dianqi, MA Xinhao, YANG Zhimei, MEI Chugang, ZAN Linsen. Bioinformatics Analysis of PAI1 Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Adipocytes in Qinchuan Beef Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1729-1741.

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Bioinformatics Analysis of PAI1 Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Adipocytes in Qinchuan Beef Cattle

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