Zfp422在猪品种间骨骼肌胚胎早期差异表达原因及对成肌分化影响的研究

Skeletal muscle development has long been a research focus in animal genetics and breeding field. In our previous study, RNA-seq of longissimus dorsi muscle from two pig breeds differing in meat production, Landrace and Lantang, during 35 days-post-coitus~180 days-post-natum were analyzed. Based on the criteria of significantly differential expression level in embryonic stages and low expression level after birth, we selected Zinc Finger Protein 422 (Zfp422) as candidate gene to regulate porcine myogenesis. Then, we proved the myogenesis regulatory function of Zfp422 through its target gene Epha7 in vitro. In this project, we plan to extract porcine embryonic fibroblast cells from LR and LT embryos to validate Zfp422’s function on myogenesis. We also plan to analyze promoter region of Zfp422, identify the upstream regulatory factor and validate its regulation with Zfp422 to study the reason of distinct Zfp422 expression between LR and LT’s skeletal muscle during early embryonic stages. Moreover, by utilizing Wuzhishan pig with lower muscle mass and Zfp422-sCKO mouse constructed in our previous study by CRISPR-Cas9 and Cre-loxp system, we aim to identify whether Zfp422 expression distinction will lead to embryonic myofiber number and adult muscle mass distinctions. In summary, this project will provide theoretical basis on understanding the regulatory mechanisms involving in different myogenesis processes of pigs differing in meat production.

骨骼肌的生长发育一直是动物遗传育种领域关注的重点。课题组前期对产肉量差异较大的长白与蓝塘猪胚胎期35至出生后180天背最长肌进行转录组分析，发现Zinc Finger Protein 422（Zfp422）在胚胎期品种间显著差异而出生后几乎不表达。最近我们在Zfp422-/- C2C12 细胞中证实Zfp422通过靶基因Epha7调节成肌发育过程，提示其可能对猪种间差异的成肌过程发挥调控作用。本项目拟在猪胚胎成纤维细胞中验证Zfp422对成肌细胞分化的作用，筛选验证Zfp422的上游调控因子，解析Zfp422在猪种间胚胎期骨骼肌中差异表达的原因，并利用产肉量递减猪种模型和可诱导型肌肉特异敲除Zfp422的基因编辑鼠探索胚胎期Zfp422的差异表达是否影响胚胎期肌纤维数量，进而造成出生后瘦肉量差异。本项目的实施将为不同猪种肌肉发育的差异调控机制和今后利用该基因提高地方猪产肉量提供理论依据。

本项目为阐明产肉量不同猪种胚胎早期骨骼肌中 Zfp422表达差异的原因及这种表达差异对胚胎期肌纤维数量及出生后产肉量的影响这两个核心问题，进行了探索。主要针对Zfp422在猪胚成肌细胞、小鼠C2C12细胞和Zfp422-scKO小鼠肌肉损伤修复及肌卫星细胞分化中的影响，并对该基因上下游调控通路进行了研究。项目研究结果明确了Zfp422在猪胚成肌细胞、小鼠C2C12细胞、Zfp422-KO C2C12细胞和Zfp422-scKO小鼠肌肉损伤修复及肌卫星细胞分化过程中发挥了关键作用，同时研究结果还发现，在猪胚成肌细胞中，Zfp422受到MyoG基因的调控来促进成肌细胞分化。本项目的实施为不同猪种肌肉发育的差异调控机制和今后利用该基因提高地方猪产肉量提供理论依据。