猪骨骼肌MyHC-Ⅱb基因上调表达的分子机制  

Molecular Mechanism of Up-Regulating the Expression of Porcine Myhc-IIb Gene in Skeletal Muscle

作　　者：张琪[1] 杜胭脂 王鹏飞[1] 李艳平[1] 王力圆[1] 李毅[1] 孙亿[1] 姜运良[1] 

机构地区：[1]山东农业大学动物科技学院&动物医学院,泰安271018

出　　处：《中国科学：生命科学》2015年第1期35-42,共8页Scientia Sinica(Vitae)

基　　金：转基因生物新品种培育科技重大专项(批准号:2011ZX08009-004);山东省农业良种工程(2013);山东省科技发展计划(批准号:2012GXG11308)资助项目

摘　　要：哺乳动物骨骼肌由各种不同类型的肌纤维镶嵌而成,不同类型肌球蛋白重链的表达是造成不同类型肌纤维的主要原因.目前已知的肌球蛋白重链家族包含8种亚型,其中长白猪骨骼肌My HC-Ⅱb的表达量显著高于中国地方猪,然而造成这种差异的分子机制未见报道.本研究用荧光定量PCR证明了长白猪背最长肌中My HC-Ⅱb m RNA的表达量显著高于莱芜猪(P=0.013).删除实验结果表明,从转录起始位点上游-1024 bp删除到-187 bp之后,My HC-Ⅱb表达量显著下降,分析发现,在这段启动子区域内存在3个E-box序列;分别突变这3个E-box序列后,My HC-Ⅱb启动子驱动的荧光素酶活性显著下降(P=0.036).另外,在My HC-Ⅱb上游启动子区?1398 bp处发现一个G>T的突变,所检测的64头莱芜猪在该位点全部为GG型,65头长白猪中13头为GG型,16头为TT型,36头为GT型.在C2C12细胞系中的转染实验结果显示,G突变为T之后有增加My HC-Ⅱb表达的趋势.Western blot的结果表明,转录因子Myo D在两猪种间表达差异不显著(P=0.136),而Myf-5在长白猪中的表达量极显著高于其在莱芜猪中的表达量(P=0.0036).这些数据表明,Myf-5是造成猪My HC-Ⅱb基因m RNA上调表达的重要因素之一.The mammalian skeletal muscle is embedded by different types of muscle fibers, mainly determined by the expression of different types of myosin heavy chain. Among the eight subtypes in the family of myosin heavy chain, both the mRNA and protein expression of MyHC-IIb were significantly higher in the skeletal muscle of Landrace pigs than in Chinese indigenous pigs. However, the mechanism underlying such differences remains largely unknown. In this study, we firstly compared the mRNA expression of MyHC-IIb in the longissimus dorsi muscle collected from Laiwu and Landrace pigs, and found that the expression level of MyHC-Ⅱb mRNA in Landrace is significantly higher than that in Laiwu pigs （P=0.013）. By transfection and luciferase assay, we found that deletion of the DNA fragment from -1024 to -187 bp in the promoter region and mutation of either of the three E-boxes in this region significantly decreased promoter activity （P）. In addition, a mutation （G〉T） was found at the site of -1398 bp in the upstream promoter region of porcine MyHC-Ⅱb gene, and population genetics analysis in Laiwu （n=64） and Landrace （n=65） pigs revealed that GG was the only genotype detected in Laiwu pigs, while in Landrace pigs, three genotypes of GG （n=13）, TT （n=16） and GT （n=36） were present. Allele T has a trend to increase MyHC-Ⅱb expression as tested in C2C12 cell line. Western blotting showed that the expressions of MyoD in two pig breeds were not significantly different （P=0.136）; however, the expressions of Myf-5 in Landrace were significantly higher than that in Laiwu pigs （P=0.0036）. These data suggest an important role of Myf-5 in up-regulating the transcription of porcine MyHC-Ⅱb gene.

关 键 词：猪 MyHC-Ⅱb 上调 启动子 Myf-5 

分 类 号：Q78[生物学—分子生物学]