湖羊PSMB9基因克隆、序列分析及对成肌细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2025.01.001

摘要/Abstract

摘要： 【目的】探讨湖羊蛋白酶体亚基β9(proteasome 20S subunit beta 9,PSMB9)基因编码区、启动子区序列特征、转录调控机制及其对成肌细胞增殖的影响。【方法】采用克隆测序法获得湖羊PSMB9基因编码区序列,根据绵羊PSMB9基因组的定位确定其启动子区大小,并通过生物学软件分析PSMB9基因编码区和启动子区序列特性,采用Mega 5.0中的邻接法(Neighbor-Joining)构建基于PSMB9氨基酸序列的系统进化树;采用实时荧光定量PCR鉴定PSMB9基因在湖羊不同组织中的表达谱;利用双酶切法构建湖羊PSMB9基因过表达载体,采用CCK-8试剂盒检测细胞的增殖能力,通过实时荧光定量PCR检测PSMB9基因在C2C12细胞系中的过表达效果和增殖基因PCNA的表达水平。【结果】湖羊PSMB9基因编码区长660 bp,编码219个氨基酸残基,其核苷酸序列和氨基酸序列与哺乳动物(如人、牛、猪和小鼠)的相似性较高,PSMB9氨基酸中活性位点和β亚基相互作用位点均高度保守;系统进化树显示,湖羊与牛先聚在一起,再与猪、人和小鼠聚在一起,说明PSMB9在哺乳动物中相对保守。PSMB9基因启动子区含有1个CpG岛、2个GC-box(CCGCCC)和2个E-box(CANNTG),且存在SP1、KLF4、STAT3、YY1、CREB1等多种转录因子潜在结合位点。组织表达谱显示,PSMB9基因在湖羊各组织中广泛表达,其中在脾脏中表达量最高,肌肉次之。与对照组相比,在C2C12细胞系中过表达PSMB9基因后极显著提升了细胞的增殖能力(P＜0.01),增殖标志基因PCNA表达水平极显著上调(P＜0.01)。【结论】本研究成功克隆获得了湖羊PSMB9基因序列,该基因在哺乳动物中高度保守,启动子区含有重要调控元件、CpG岛和转录因子潜在结合位点。PSMB9基因在湖羊脾脏和肌肉组织中高表达,PSMB9基因过表达可促进成肌细胞的增殖。研究结果为进一步阐明PSMB9基因调控湖羊肌肉发育的分子机制提供依据。

关键词: 湖羊; PSMB9基因; 克隆; 组织表达谱; C2C12细胞系; 细胞增殖

Abstract: 【Objective】 This study was aimed to investigate the sequence characteristics of coding region and promoter region of the proteasome 20S subunit beta 9 (PSMB9) gene in Hu sheep,and analyze its potential transcriptional regulation mechanism and its impact on myoblast cell proliferation. 【Method】 The CDS region of PSMB9 gene in Hu sheep was obtained by cloning and sequencing,the promoter region was determined by genome position in sheep,and the characteristics of CDS and promoter region were analyzed by bioinformatics method.The phylogenetic tree based on the amino acid sequence of PSMB9 was constructed using Neighbor-Joining (NJ) method of Mega 5.0 software.The tissue expression profile of PSMB9 gene in Hu sheep was detected by Real-time quantitative PCR.The overexpression vector of PSMB9 gene in Hu sheep was constructed by double enzyme digestion.CCK-8 kit were determined to detect the proliferative capacity,Real-time quantitative PCR was used to detect the overexpression effect of PSMB9 gene in C2C12 cells and the expression of proliferation gene PCNA. 【Result】 The results showed that the CDS region of PSMB9 gene in Hu sheep was 660 bp,encoding 219 amino acid residues,and the similarity of nucleotide and amino acid sequences with mammals including Homo sapiens,Bos taurus,Sus scrofa and Mus musculus were high.The active sites and beta subunit interaction sites of PSMB9 were highly conserved.Phylogenetic tree analysis found that Hu sheep first clustered with Bos taurus,and then clustered with Sus scrofa,Homo sapiens and Mus musculus,indicating that PSMB9 was relatively conserved in mammals.PSMB9 gene promoter contained one CpG island,two GC-boxes (CCGCCC) and two E-boxes (CANNTG),as well as potential binding sites for various transcription factors such as SP1,KLF4,STAT3,YY1 and CREB1.Tissue expression pattern showed PSMB9 gene was widely expressed in various tissues of Hu sheep,especially highly expressed in spleen and followed by muscle.Compared with control group,overexpression of PSMB9 gene in C2C12 cell lines could extremely significantly improve the proliferation ability of cells (P＜0.01),the expression of proliferation gene PCNA was extremely significantly upregulated (P＜0.01). 【Conclusion】 The sequence of PSMB9 gene in Hu sheep was successfully cloned in this study,PSMB9 gene was highly conserved in mammals,the promoter region of PSMB9 gene contained important regulatory elements,CpG island and potential binding sites of transcription factors.PSMB9 gene was highly expressed in spleen and muscle of Hu sheep,and overexpression of PSMB9 gene could promote the proliferation of myoblasts.The results provided a basis for further elucidating the molecular mechanism of PSMB9 gene regulation of muscle development in Hu sheep.

Key words: Hu sheep; PSMB9 gene; cloning; tissue expression pattern; C2C12 cell lines; cell proliferation

中图分类号:

S826
Q78

谢蓓伊庭, 王悦, 孟春花, 钱勇, 张俊, 张建丽, 王慧利, 曹少先, 李隐侠. 湖羊PSMB9基因克隆、序列分析及对成肌细胞增殖的影响[J]. 中国畜牧兽医, 2025, 52(1): 1-12.

XIE Beiyiting, WANG Yue, MENG Chunhua, QIAN Yong, ZHANG Jun, ZHANG Jianli, WANG Huili, CAO Shaoxian, LI Yinxia. Cloning and Sequence Analysis of PSMB9 Gene in Hu Sheep and Its Effect on Myoblast Proliferation[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(1): 1-12.

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