关岭牛MyoD1与MSTN基因相互表达调控  

Mutual expression regulation of MyoD1 and MSTN genes in Guanling cattle

作　　者：田念念 李雄[1,2] 黄勇 石鹏飞[1,2] 孙金魁 许厚强 TIAN Nian-nian;LI Xiong;HUANG Yong;SHI Peng-fei;SUN Jin-kui;XU Hou-qiang(Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountains Region of Ministry of Education,Guizhou University/Key Laboratory of Animal Genetics,Breeding and Reproduce in Guizhou Province,Guiyang 550025,China;College of Animal Sciences,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵州贵阳550025 [2]贵州大学动物科学学院,贵州贵阳550025

出　　处：《生物技术》2021年第5期417-424,共8页Biotechnology

基　　金：国家科技支撑计划项目(2015BAD03B02-3)。

摘　　要：[目的]旨在验证牛MyoD1与MSTN在表达调控上的相互作用。[方法]首先,以构建的p EGFP-N3-MyoD1和p EGFP-N3-MSTN过表达载体,分别在两组关岭牛原代成肌细胞中过表达MyoD1和MSTN,细胞转染24 h后,qRT-PCR检测并分析各处理组细胞中MyoD1和MSTN相比表达情况。其次,以海肾荧光载体pRL-TK为内参质粒,在转染p GL-3-MSTN-pro质粒的关岭牛原代成肌细胞中过表达MyoD1,在转染p GL-3-MyoD1-pro质粒的关岭牛原代成肌细胞中过表达MSTN,共转染36 h后,双荧光素酶试剂盒检测分析各组细胞中相对荧光素酶活性变化。[结果]过表达MyoD1组细胞中MyoD1相对表达量相比对照组上调至8 283.987±2 337.534(P=0.004),MSTN的相对表达量上调至7.849±1.089 (P=0.000 4);而MSTN过表达组细胞中MSTN相对表达量相比对照组上调至121 131.802±12 474.046(P=0.004),MyoD1基因的相对表达量下调至0.103±0.016(P=0.000 1)。进一步试验中,过表达MyoD1组细胞中相对荧光素酶活性变化相比对照组从1.131±0.051上调至3.030±0.212(P=0.0001);而过表达MSTN组细胞中相对荧光素酶活性变化相比对照组从1.797±0.136下调至0.543±0.092(P=0.000 2)。[结论]MyoD1的过量表达(P=0.004),会导致MSTN表达显著上调(P=0.000 4);而MSTN的过量表达(P=0.004),可负向调节MyoD1表达(P=0.000 1)。此外MyoD1的过表达会增强MSTN启动子活性(P=0.000 1);而MSTN的过表达会降低MyoD1启动子活性(P=0.000 2)。综上,MyoD1可正向调控MSTN表达,而MSTN也对MyoD1表达起负反馈调节作用。[Objective]To verify the interaction between bovine MyoD1 and MSTN in expression regulation.[Method]Firstly,the constructed p EGFP-N3-MyoD1 and p EGFP-N3-MSTN overexpression vectors were used to overexpress MyoD1 and MSTN in Guanling cattle primary myoblast cells in two groups respectively. 24 hours after transfection,qRT-PCR was used to detect and analyze the relative expression of MyoD1 and MSTN in cells of each group. Secondly,p GL-TK was used as the reference plasmid to overexpress MyoD1 in the Guanling cattle primary myoblast cells transfected with p GL-3-MSTN-pro,and MSTN was overexpressed in the cells transfected with p GL-3-MyoD1-pro. 36 hours after co-transfection,the relative luciferase activity changes in each group were detected and analyzed with Dual-GloLuciferase Assay System.[Result]Compared with the control group,the relative expression of MyoD1 in the overexpressed MyoD1 group was up-regulated to 8 283. 987 ±2 337. 534( P = 0. 004),then the relative expression of MSTN gene was up-regulated to 7. 849 ± 1. 089( P = 0. 000 4). And the relative expression of MSTN in the MSTN overexpression group was up-regulated to 121 131. 802 ± 12 474. 046( P =0. 004),then the relative expression of MyoD1 was down-regulated to 0. 103 ± 0. 016( P = 0. 000 1). In further experiments,the relative luciferase activity fold change in MyoD1 overexpressed cells were increased from 1. 131 ± 0. 051 to 3. 030 ± 0. 212( P = 0. 000 1) compared with the control cells. relative luciferase activity fold change in the MSTN overexpressed group was decreased from 1. 797 ± 0. 136 to 0. 543 ± 0. 092( P = 0. 000 2).[Conclusion]Overexpression of MyoD1( P = 0. 004) resulted in significantly upregulated expression of MSTN( P = 0. 000 4). However,the overexpression of MSTN( P = 0. 004) could negatively regulate the expression of MyoD1( P = 0. 000 1). In addition,the overexpression of MyoD1 enhanced the activity of MSTN promoter( P = 0. 000 1). However,the overexpression of MSTN could decrease the activity of MyoD1 promoter( P = 0

关 键 词：关岭牛 MyoD1 MSTN 过表达 双荧光素酶 启动子 

分 类 号：S831.2[农业科学—畜牧学]