山羊CSRP3基因对成肌细胞分化的功能研究

doi:10.3864/j.issn.0578-1752.2026.09.012

摘要/Abstract

摘要：

【目的】骨骼肌的生长发育是影响羊肉品质的重要因素之一。通过克隆山羊CSRP3（cysteine and glycine rich protein 3）的编码区（coding sequence, CDS）序列，并进行生物信息学分析；探究CSRP3在山羊不同组织、成肌细胞不同分化阶段的表达规律；明确其对成肌细胞分化的调控作用。【方法】采集山羊心脏、肝脏、脾脏、肺、肾脏、背最长肌组织，Trizol法提取组织总 RNA 并反转录为 cDNA；以背最长肌组织cDNA为模板，利用RT-PCR扩增CSRP3的CDS序列，运用在线软件进行生物信息学分析；分别以山羊不同组织、不同分化阶段的成肌细胞的cDNA为模板，通过实时荧光定量PCR（qPCR）检测山羊CSRP3基因的mRNA相对表达量；通过过表达载体构建、siRNA 合成和细胞转染试验，利用 qPCR、免疫荧光染色（immunofluorescence，IF）检测过表达和干扰山羊CSRP3后成肌细胞分化标志基因的mRNA相对表达量及肌管的形成情况。【结果】山羊CSRP3全长1 103 bp，CDS序列长 585 bp，系统进化树结果显示，山羊CSRP3与绵羊、牛的亲缘关系最近，提示该基因在近缘物种间具有较高的保守性；山羊CSRP3编码194个氨基酸，其编码的蛋白为亲水性、稳定的碱性蛋白；蛋白互作关系分析表明，山羊CSRP3可能通过与肌肉功能相关蛋白互作参与肌肉生长发育；CSRP3在山羊背最长肌和心肌中的mRNA相对表达量显著高于其他组织（P < 0.05），在成肌细胞分化初期表达量较低，随着分化时间增加mRNA相对表达量逐渐增加，在分化第3天达到峰值（P< 0.01）后下降，呈现出先升高后下降的动态变化，提示山羊CSRP3可能在成肌细胞由增殖向分化的转变中发挥重要调控作用；过表达山羊CSRP3显著提高成肌细胞分化标志基因的mRNA相对表达量和肌管形成，促进成肌细胞分化；反之，干扰山羊CSRP3则显著降低成肌细胞分化标志基因的mRNA相对表达量和肌管形成，抑制成肌细胞的分化能力。【结论】成功克隆山羊CSRP3的CDS序列（克隆序列长603 bp，其中CDS区长度为585 bp），明确其序列特征与理化性质；成功绘制了山羊CSRP3的组织表达谱和时序表达谱；明确了CSRP3对山羊成肌细胞分化具有正向调控作用。初步表明，CSRP3对肉用山羊肌肉生长发育中具有重要生物学功能。

关键词: 山羊, CSRP3, 组织表达, 时序表达, 成肌分化

Abstract:

【Objective】Skeletal muscle growth and development is one of the important factors affecting meat quality of goat. This study aimed to clone the coding sequence (CDS) of the goat CSRP3 gene (Cysteine and Glycine Rich Protein 3), conduct bioinformatics analysis using the online software, investigate the expression patterns of the goat CSRP3 gene in different tissues and at different differentiation stages of goat myoblasts, and clarify the regulatory role of this gene in myoblast differentiation.【Method】Tissues including the heart, liver, spleen, lung, kidney, and longissimus dorsi muscle were collected from goats. Total RNA was extracted from these tissues using the Trizol method and was reverse-transcribed to cDNA. Using the cDNA from the longissimus dorsi muscle as the template, RT-PCR was performed to amplify the CDS region of the goat CSRP3 gene, and online software was used for bioinformatics analysis. cDNA from different goat tissues and myoblasts at different differentiation stages was used as templates to detect the CSRP3 gene expression level by quantitative Real-Time PCR (qPCR). Through the construction of an overexpression vector, siRNA synthesis, and cell transfection experiments, qPCR and immunofluorescence staining were employed to determine the relative mRNA expression level of myoblast differentiation marker genes and the formation of myotubes after overexpression or interference of the goat CSRP3 gene.【Result】The full-length of the goat CSRP3 gene is 1 103 bp, with a CDS sequence of 585 bp. Phylogenetic tree analysis showed that the goat CSRP3 gene has the closest genetic relationship with sheep and cattle, indicating high conservation of this gene among closely related species. The goat CSRP3 gene encodes 194 amino acids, and the encoded protein is a hydrophilic and stable basic protein. Protein-protein interaction analysis revealed that the goat CSRP3 protein may be involved in muscle growth and development through interactions with muscle function-related proteins. The relative mRNA expression level of the CSRP3 gene in goat longissimus dorsi muscle and heart was significantly higher than that in other tissues (P < 0.05). During myoblast differentiation, the goat CSRP3 gene expression was low at the initial stage, increased gradually with differentiation time, peaked on day 3 (P < 0.01), and then decreased, showing a dynamic change of first increasing and then decreasing. This suggested that the goat CSRP3 gene may play an important regulatory role in the transition of myoblasts from proliferation to differentiation. Overexpression of the goat CSRP3 gene significantly increased the relative mRNA expression levels of myoblast differentiation marker genes and promoted myotube formation, indicating myoblast differentiation. Conversely, interference with the CSRP3 gene significantly reduced the relative mRNA expression levels of myoblast differentiation marker genes and inhibited myotube formation, impairing the differentiation ability of myoblasts.【Conclusion】The CDS of CSRP3 was successfully cloned (the cloned sequence is 603 bp in length, including a 585 bp CDS region), and its sequence characteristics and physicochemical properties were clarified. The CSRP3 tissue and temporal expression profiles in goats were established. It was confirmed that the CSRP3 gene exerts a positive regulatory effect on goat myoblast differentiation. These results preliminarily indicate that the CSRP3 gene plays an important biological function in the muscle growth and development of meat goats.

Key words: goat, CSRP3 gene, tissue expression, temporal expression, myoblast differentiation

马辉风, 文淑良, 刘莞颜, 林亚秋, 李鑫, 刘珂含, 朱荟颖, 薛福来, 邢佳妮. 山羊CSRP3基因对成肌细胞分化的功能研究[J]. 中国农业科学, 2026, 59(9): 2002-2015.

MA HuiFeng, WEN ShuLiang, LIU WanYan, LIN YaQiu, LI Xin, LIU KeHan, ZHU HuiYing, XUE FuLai, XING JiaNi. Functional Study of Goat CSRP3 Gene on Myoblast Differentiation[J]. Scientia Agricultura Sinica, 2026, 59(9): 2002-2015.

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基因 Gene	序列 Sequence	退火温度 Tm (℃)	产物长度 Product length (bp)	用途 Uses
CSRP3	F: tagCTCGAGatgccaaactggggtggag R: tacGGATCCtcaatctttcttttccacttgg	59	603	RT-PCR
CSRP3	F: CCGTCTACCATGCGGAAGAA R: GATCTCTGACTCGTGAGCGG	60	122	qPCR
UXT	F: GCAAGTGGATTTGGGCTGTAAC R: ATGGAGTCCTTGGTGAGGTTGT	60	180	qPCR
MYHC	F: CTCTTCCCGCTTTGGTAAGTT R: CAGGAGCATTTCGATTAGATCCG	60	187	qPCR
MYOG	F: GCAGGCTCAAGAAAGTGAATGA R: TAGGCGCTCAATGTACTGGAT	60	122	qPCR
MYOD1	F: GTGCAAACGCAAGACGACTA R: GCTGGTTTGGGTTGCTAGAC	59	128	qPCR

软件Software	网址 Websites	功能预测Function prediction
ProParam	https://web.expasy.org/protparam/	理化性质physicochemical properties
ProScale	https://web.expasy.org/protscale/	亲/疏水性hydrophilicity/hydrophobicity
SOPMA	https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.plpage=/NPSA/npsa_sopma.html	二级结构secondary structure
SWISS-MODEL	https://swissmodel.expasy.org/	三级结构tertiary structure
STRING	http://string-db.org/	蛋白互作protein-protein interaction

基因 Gene	正义链5′—3′ Sense 5′-3′	反义链5′—3′ Antisense 5′-3′
CSRP3-231	AGUGCAAUGGAAGGAGUUUTT	AAACUCCUUCCAUUGCACUTT
CSRP3-311	GCUCACGAGUCAGAGAUCUTT	AGAUCUCUGACUCGUGAGCTT
CSRP3-521	CCUCGAUGCGGAAAGUCAGTT	CUGACUUUCCGCAUCGAGGTT
CSRP3-612	GGAAGAGUCUAGAGUCCACTT	GUGGACUCUAGACUCUUCCTT
Negative Control FAM	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT