猪CFL2基因表达对骨骼肌肌纤维的影响及分子作用机制

Meat quality is determined by types of skeletal muscle fibers which could change each other. The important hole for pig breeding of meat quality trait is to seek molecular markers or major genes. We found in our previous research that the high or low expression of pig cofilin 2 (muscle-type cofilin or cofilin 2, CFL2) could influence the expression of muscle fibers and myosin heavy chain (MyHC) isoforms significantly. Further study found that expression levels of CFL2 were significantly positive or nagative associated with MyHC isoforms, diameter of muscle fibers, shear value and pH during the postnatal development in native and imported pigs. However, the mechanism of influence and association of CFL2 expression on/and muscle fibers is still not clarified. Based on our previous results, the objects of this project are to acquire the features of promotor of swine CFL2 by the way of promoter length deficiency, analyze the relattionship between important common key control components (CFL2, MLCK and MEF2) from four signal pathways controlling the synthesis of myofibril composition substances with high/low expression of CFL2, MLCK or MEF2 in C2C12 cell lines, CFL2 transgenic mice model, slaughter tests at a series of postnatal periods in 5 wild, native and imported pigs, co-immunoprecipi- tation (Co-IP), GST pull-down, and laser confocal. These results will reveal the mechanism of CFL2 affecting muscle fibers and MyHC isoforms and enrich the theory of muscle fibers formation. The mathematical mode between CFL2 and muscle fibers and MyHC isotypes will be built and play the basis for application in production.

骨骼肌肌纤维类型可以相互转化并决定猪肉品质，因此寻找调控肌纤维的分子标记/主效基因成为猪育种重要突破口。我们前期研究发现，猪骨骼肌丝切蛋白2（CFL2）高/低表达显著影响肌纤维总量及MyHC异构体。进一步研究发现，中外猪种CFL2表达量与MyHC异构体、肌纤维直径、剪切值等呈显著正/负相关，但这种影响/相关的分子作用机制仍不清楚。本课题在前期工作基础上，通过启动子长度缺失方法，获得猪CFL2基因启动子特点；通过基因在C2C12细胞株中高/低表达、转基因小鼠模型、5种中外猪种和商品猪生后表达，以及免疫共沉淀、pull-down和激光共聚焦探索影响肌原纤维组成成分的4个信号通路共同关键控制成分（CFL2、MLCK和MEF2）的相互关系，揭示CFL2影响肌纤维和MyHC异构体的分子作用机制，丰富了肌纤维调控的基础理论；将建立CFL2与肌纤维和MyHC异构体的相关数学模型，为生产应用奠定基础。

背景：前期研究发现猪骨骼肌丝切蛋白2（CFL2）显著影响肌纤维总量及MyHC异构体，但作用机理不清。研究内容：通过染色体步移法和启动子长度缺失法获得猪CFL2基因5'上游序列及启动子活性区域。通过CFL2、MEF2C和skMLCK分别在C2C12细胞高/低表达、CFL2转基因小鼠、4种猪生后不同阶段对肌纤维及信号通路的检测，建立CFL2与肌纤维和MyHC异构体表达的相关数学模型，探讨CFL2作用机制、CFL2与MEF2C和skMLCK关系。通过免疫共沉淀、质谱和WB探索与CFL2和MEF2C互作的蛋白。结果：获得2.6kb的CFL2基因5'上游序列及启动子，启动子活性区域为-1502bp~-1317bp。所有10个独立实验数据综合分析后发现，CFL2与肌纤维MyHC总量、MyHC2x、MyHC2b、MLC和α-actin均呈极显著（p<0.01）正相关关系，并建立了相关数学模型；CFL2与MyHC2b的相关系数r=0.845，数学模型拟合度R2=0.719。CFL2与MEF2C显著相关，共同通过CaM和AMPKα2反馈调节肌纤维发育表达；而CFL2与skMLCK相关不显著，skMLCK是通过MAPKp38和RhoA信号通路反馈调节肌纤维发育。调控猪生后肉质性状主要是CaM和MAPKp38信号通路。与CFL2互作的3种蛋白是Tnnc2、Mylpf、Ndufb7；与MEF2C互作的4种蛋白是β-actin、GAPDH、LDHA、MDHM。科学意义：本项目完善了猪CFL2全基因组序列，获得了启动子活性区域；CFL2是肌纤维及亚型合成的蛋白标记，尤其是MyHC2b的蛋白标记，并建立了相关数学模型；获得了CFL2影响肌纤维和MyHC异构体的分子作用机制，明确了CFL2与MEF2C和skMLCK的关系，丰富了肌纤维信号通路调控的基础理论，为后续的分子标记辅助育种生产应用奠定了基础。