Cloning, Bioinformatics and Tissue Expression Analysis of CSRP3 Gene in Hezuo Pig (Sus scrofa)
LI Xiao1, YANG Quan1, NIU Pin-Yuan1, SONG Ke-Lin1, YAN Zun-Qiang1, GUN Shuang-Bao1,2,*, YANG Jiao-Jiao1,2,*
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Province Modern Pig Engineering Technology Innovate Center, Lanzhou 730070, China
Abstract：The cysteine and glycine rich protein 3 (CSRP3) gene, as a key regulator of animal muscle fiber development, plays a core role in muscle growth and functional maintenance. Through using PCR amplification and sequencing, the full-length CDS sequence of CSRP3 gene in Hezuo pig was obtained. Bioinformatics analysis software was used to perform similarity comparisons, phylogenetic tree construction, and domain prediction analysis on the gene sequence. Additionally, qRT-PCR was employed to detect the expression levels of the CSRP3 gene in 11 tissues of Hezuo pigs, including the heart, liver, spleen, and longissimus dorsi muscle, The results showed that the CDS of CSRP3 gene of Hezuo pig was 585 bp and encoded 194 amino acids, there were 4 base mutations, all of them were synonymous mutations. The homologous sequence comparison and phylogenetic tree analysis showed that Hezuo pig had the closest relationship with Duroc and the most distant relationship with Gallus gallus; the molecular weight of 20.94 kD, the isoelectric point (pI) was 8.89, and the coefficient was 39.11, protein with fewer hydrophobic amino acid residues than hydrophilic ones. It had neither a transmembrane structure nor a signal peptide; analysis of its expression characteristics in different tissues showed that this gene had the highest expression level in the heart of Hezuo pigs and the lowest in the liver. This study provides a reference for further functional studies of the CSRP3 gene in Hezuo pigs.
[1] 白佳灵, 王会, 柴志欣, 等. 2020. 牦牛CSRP3基因的克隆及组织表达分析[J]. 生物技术通报, 36(6): 200-207. (Bai J L, Wang H, Chai Z X, et al.2020. Cloning and tissue expression analysis of CSRP3 in yak[J]. Biotechnology Bulletin, 36(6): 200-207. [2] 蔡原, 赵生国. 2012. 合作猪肌肉氨基酸含量测定及营养价值评价[J]. 中兽医医药杂志, 31(05): 17-19. (Cai Y, Zhao S G.2012. Determination of amino acid content and nutritional value evaluation of Hezuo pig muscle[J]. Journal of Traditional Chinese Veterinary Medicine, 31(05): 17-19. [3] 陈云, 潘鹏丞, 陈钊, 等. 2022. 陆川猪CSRP3基因克隆及其组织表达差异分析[J]. 南方农业学报, 53(04): 977-984. (Chen Y, Pan P C, Chen Z, et al.2022. Cloning and differential expression analysis of CSRP3 gene in Luchuan pig[J]. Journal of Southern Agriculture, 53(4): 977-984. [4] 刘孟洲, 王玉涛. 2006. 合作猪种质特性的研究[J]. 中国畜牧杂志, 42(13): 6-8. (Liu M Z, Wang Y T.2006. Study on breed characteristic of Hezuo pig[J]. Chinese Journal of Animal Science, 42 (13): 6-8.) S828.8+9 [5] 刘雪, 徐琳娜. 2024. 合作猪种质资源保护利用现状、问题及对策[J]. 甘肃畜牧兽医, 54(03): 17-21. (Liu X, Xu L N.2024. Present situation, problems and countermeasures of protection and utilization of Hezuo pig germplasm resources[J]. Gansu Animal Husbandry and Veterinary Medicine, 54(03): 17-21. [6] 李歆怡, 王福彬, 黄纯, 等. 2023. 大通牦牛PDGFD基因克隆、生物信息学及组织表达谱分析[J]. 农业生物技术学报, 31(03): 518-529. (Li X Y, Wang F B, Huang C, et al.2023. Cloning, bioinformatics and tissue expression profile analysis of PDGFD gene in Yak (Bos mutus)[J].Journal of Agricultural Biotechnology, 31(03): 518-529. [7] 单艳菊, 姬改革, 邹剑敏, 等. 2020. 鸡CSRP3基因编码区SNPs及其与肌纤维性状的相关分析[J]. 中国家禽, 42(1): 7-11. (Shan Y J, Ji G G, Zou J M, et al.2020. Polymorphisms of coding region in CSRP3 gene and its association with muscle fiber traits in chickens[J]. China Poultry, 42(1): 7-11. [8] 单艳菊, 姬改革, 章明, 等. 2023. CSRP3基因在鸡胚胎期胸肌、成肌细胞中的表达及其与胸肌质量的相关性研究[J]. 中国畜牧杂志, 59(01): 131-135. (Shan Y J, Ji G G, Zhang M, et al.2023. Expression of CSRP3 gene in embryonic chicken pectoralis muscle and chicken myoblasts and its correlation with chest muscle mass[J]. Chinese Journal of Animal Science, 59(01): 131-135. [9] 商鹏. 2017. 基于胚胎肌肉组织转录组和蛋白质组数据鉴定猪生长性状相关基因[D]. 博士学位论文, 中国农业大学, 导师: 吴常信, 张浩, pp. 72-75. (Shang P.2017. Identification of pig growth trait related genes based on transcriptome and proteomic data of embryonic muscle tissue[D]. Thesis for Ph. D., China Agricultural University, Supervisor: Wu C X, Zhang H, pp. 72-75.) [10] 王晴, 马瑜锴, 韩若冰, 等. 2025. 梅花鹿CSRP3基因克隆、生物信息学分析及其在肌肉组织中的表达分析[J]. 中国畜牧兽医, 52(02): 740-748. (Wang Q, Ma Y K, Han R B, et al.2025. Cloning,bioinformatics analysis and expression analysis of CSRP3 gene in muscle tissues of sika deer[J]. China Animal Husbandry & Veterinary Medicine, 52(02): 740-748. [11] 徐学文. 2007. 建立在猪骨骼肌差异表达EST基础上的新基因cDNA克隆及其功能研究[D]. 博士学位论文, 华中农业大学, 导师: 刘榜, pp. 49-53. (Xu X W.2007. cDNA cloning based on differential expressed EST in porcine skeletal muscle and functional characterization of novel genes[D]. Thesis for Ph. D., Huazhong Agricultural University, Supervisor: Liu B, pp. 49-53.) [12] 闫尊强, 鲁亚伟, 滚双宝, 等. 2025. 合作猪NTF3基因克隆、生物信息学分析及组织表达谱构建[J]. 西北农业学报, 34(01): 1-9. (Yan Z Q, Lu Y W, Gun S B, et al.2025. Cloning,bioinformatics analysis and tissue expression profile construction of NTF3 gene in Hezuo pig[J]. Acta Agriculturae Boreali-occidentalis Sinica, 34(01): 1-9. [13] 张海峰, 余淑琴, 马永国, 等. 2024. 岷县蕨麻猪种质资源保护现状与利用[J]. 猪业科学, 41(12): 109-111. (Zhang H F, Yu S Q, Ma Y G, et al.2024. The current status and utilization of germplasm resources protection of Min county fern and hemp pigs[J]. Swine Industry Science, 41(12): 109-111.) [14] Arber S, Hunter J J, Ross J, et al.1997. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure[J]. Cell, 88(3): 393-403. [15] Barash I A, Mathew L, Lahey M, et al.2005. Muscle LIM protein plays both structural and functional roles in skeletal muscle[J]. American Journal of Physiology-Cell Physiology, 289(5): C1312-C1320. [16] Chauhan P K, Sowdhamini R.2022. LIM domain-wide comprehensive virtual mutagenesis provides structural rationale for cardiomyopathy mutations in CSRP3[J]. Scientific Reports, 12(1): 3562. [17] Chauhan P K, Sowdhamini R.2023. Computational analysis of the flexibility in the disordered linker region connecting LIM domains in cysteine-glycine-rich protein[J]. Frontiers in Genetics, 14: 1134509. [18] Flick M J, Konieczny S F.2000. The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin[J]. Journal of Cell Science, 113(9): 1553-1564. [19] Geier C, Gehmlich K, Ehler E, et al.2008. Beyond the sarcomere CSRP3 mutations cause hypertrophic cardiomyopathy[J]. Human Molecular Genetics, 17(18): 2753-2765. [20] He H, Zhang H L, Li Z X, et al.2014. Expression, SNV identification, linkage disequilibrium, and combined genotype association analysis of the muscle-specific gene CSRP3 in Chinese cattle[J]. Gene, 535(1): 17-23. [21] Louis H A, Pino J D, Schmeichel K L, et al.1997. Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression[J]. The Journal of Biological Chemistry, 272(43): 27484-27491. [22] Liu G Q, Zhang C L, Wang G Z, et al.2016. Molecular cloning,characterization and tissue specificity of the expression of the ovine CSRP2 and CSRP3 genes from small-tail Han sheep (Ovis aries)[J]. Gene, 580(1): 47-57. [23] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔCt) method[J]. Methods, 25(4): 402-408. [24] Paudyal A, Dewan S, Ikie C, et al.2016. Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac functionin the transition to failure[J]. The Journal of Physiology. 594(12): 3287-305. [25] Velleman S G, Coy C S, Abasht B.2021. Effect of growth selection of broilers on breast muscle satellite cell function: Response of satellite cells to NOV, COMP, MYBP-C1, and CSRP3[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 255: 110917. [26] Weiskirchen R, Pino J D, Macalma T, et al.1995. The cysteine-rich protein family of highly related LIM domain proteins[J]. Journal of Biological Chemistry. 270(48): 28946-28954. [27] Xu X W, Qiu H F, Du Z Q, et al.2010. Porcine CSRP3: Polymorphism and association analyses with meat quality traits and comparative analyses with CSRP1 and CSRP2[J]. Molecular Biology Reports, 37(1): 451-459. [28] Zhang B, Yan Z Q, Gao Y, et al.2022. Integrated analysis of miRNA and mRNA expression profiles in testes of Landrace and Hezuo boars[J]. Frontiers in Veterinary Science, 9: 942669. [29] Zhang B, Yan Z Q, Wang P F, et al.2021. Identification and characterization of lncRNA and mRNA in testes of Landrace and Hezuo boars[J]. Animals, 11(8): 2263.
