干扰MSTN的牛骨骼肌卫星细胞实时荧光定量PCR内参基因的筛选

doi:10.16431/j.cnki.1671-7236.2020.02.008

摘要/Abstract

摘要： 为了筛选在牛骨骼肌卫星细胞（MSCs）分化前后稳定表达以及不受MSTN基因表达影响的内参基因，试验以野生组（WT）、转染干扰MSTN组（si-MSTN）和对照组（NC-MSTN）的牛MSCs作为样品，选取HMBS、B2M、GAPDH、TUBB、SDHA、18S rRNA、ACTB、RPL4、PPIA、HPRT1和YWHAZ作为候选内参基因，采用实时荧光定量PCR技术Ct值分析法对各候选内参基因的相对表达进行测定，首先利用3个独立评价软件geNorm、NormFinder和BestKeeper分别对各候选内参基因在野生组牛MSCs增殖期（GM）和分化第3天（DM3）细胞中的表达稳定性进行评价，筛选出前5个表达相对稳定的候选内参基因；然后以此为基础，利用相同的方法分析MSTN干扰组和对照组增殖期和分化第3天的细胞中上述5个内参基因的表达水平和稳定性。geNorm、NormFinder分析结果均显示，HMBS、B2M、TUBB、GAPDH和ACTB在牛MSCs分化前后表达较稳定，BestKeeper分析显示ACTB和TUBB相关系数（r）排序靠前，GAPDH、HMBS和B2M排序不是很高，但是GAPDH的SD值最小，因此选择这5个候选内参基因做后续试验；在牛MSCs MSTN干扰组和对照组增殖期和分化第3天，geNorm、NormFinder软件分析结果显示，上述5个候选内参基因中GAPDH、TUBB和B2M表达最稳定，BestKeeper分析显示TUBB相关系数排名不是最高，但其SD值最小，综合以上分析，选择TUBB作为牛MSCs干扰组和对照组增殖期和分化第3天最适内参基因。研究结果可为以后进行牛MSCs在不同生长时期以及MSTN表达被调控后基因的表达分析提供参考。

关键词: 肌肉生长抑制素(MSTN); 牛; 骨骼肌卫星细胞(MSCs); 内参基因; 稳定性

Abstract: In order to screen the reference genes that suitable for bovine skeletal muscle satellite cells (MSCs) differentiation process and not affected by MSTN gene expression,bovine MSCs from wild group (WT),MSTN interfered group (si-MSTN) and control group (NC-MSTN) were used as sample.HMBS,B2M,GAPDH,TUBB,SDHA,18S rRNA,ACTB,RPL4,PPIA,HPRT1 and YWHAZ were selected as candidate internal reference genes.The relative expression of candidate internal reference genes was determined by Real-time quantitative PCR and Ct value analysis.Firstly,the expression stability of each candidate internal reference gene in wild group (WT) bovine MSCs of proliferative (GM) and the differentiated third day after differention (DM3) cells was evaluated by three independent evaluation softwares:geNorm,NormFinder and BestKeeper,and the first five candidate reference genes with the most stable expression were screened out.Furthermore,the expression level and stability of these five internal reference genes in si-MSTN group and NC-MSTN group of GM and DM3 were analyzed by similar methods.The analysis of geNorm and NormFinder showed that the expression of HMBS,B2M,TUBB,GAPDH and ACTB genes were more stable before and after differentiation of bovine MSCs.The BestKeeper showed that the correlation coefficient (R) of ACTB and TUBB were ranked first,GAPDH,HMBS and B2M were not very high,but GAPDH had the lowest SD value,so these five candidate genes were selected for subsequent experiments.In siRNA-MSTN group and NC-MSTN group of GM and DM3,the results of geNorm and NormFinder showed that the expressions of GAPDH,TUBB and B2M in the five candidate reference genes were more stable than other.BestKeeper showed that the correlation coefficient of TUBB was not the highest,but its SD value was the lowest.Based on the above analysis,TUBB was selected as the most suitable internal reference gene in GM and DM3 of bovine MSCs siRNA-MSTN and NC-MSTN groups.The result of this study would provide reference for future research on gene expression analysis of bovine MSCs in different growth process and after MSTN expression was regulated.

Key words: MSTN; bovine; MSCs; internal reference genes; stability

中图分类号:

S813.3

盛辉, 张林林, 郭益文, 李新, 丁向彬, 郭宏. 干扰MSTN的牛骨骼肌卫星细胞实时荧光定量PCR内参基因的筛选[J]. 中国畜牧兽医, 2020, 47(2): 381-391.

SHENG Hui, ZHANG Linlin, GUO Yiwen, LI Xin, DING Xiangbin, GUO Hong. A Selection of Reference Genes for Real-Time Quantitative PCR in Bovine Skeletal Muscle Satellite Cells Interfering with MSTN[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 381-391.

参考文献

[1] 文逸凡,贺花,宋成创,等.家养动物肌肉发育相关基因的研究进展[J].基因组学与应用生物学,2018,37(7):2825-2831. WEN Y F,HE H,SONG C C,et al.Research progress of muscle development related genes in domestic animals[J].Genomics and Applied Biology,2018,37(7):2825-2831.(in Chinese)
[2] LEE S J,MCPHERRON A C.Regulation of myostatin activity and muscle growth[J].Proceedings of the National Academy of Sciences,2001,98(16):9306-9311.
[3] MCPHERRON A C,LAWLER A M,LEE S J.Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[J].Nature,1997,387(6628):83-90.
[4] YANG S P,LI X,LIU X F,et al.Parallel comparative proteomics and phosphoproteomics reveal that cattlemyostatinregulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway[J].Oncotarget,2018,9(13):11352-11370.
[5] DENG B,DING Y,WEN J H,et al.Different regulation role of myostatin in differentiating pig ADSCs and MSCs into adipocytes[J].Cell Biochemistry and Function,2012,30(2):145-150.
[6] PIRRUCCELLO-STRAUB M,JACKSON J,WAWERSIK S,et al.Blocking extracellular activation of myostatin as a strategy for treating muscle wasting[J].Scientific Reports,2018,8(1):2292.
[7] MANFREDI L H,PAULA-GOMES S,ZANON N M,et al.Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents[J].Brazilian Journal of Medical and Biological Research,2017,50(12):e6733.
[8] SABROUT E I,AGGAG S.Association of Melanocortin (MC4R) and Myostatin (MSTN) genes with carcass quality in rabbit[J].Meat Science,2018,137:67-70.
[9] MOSHER D S,QUIGNON P,BUSTAMANTE C D,et al.A mutation in the Myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs[J].PLoS Genetics,2007,3(5):e79.
[10] WANG K,OUYANG H,XIE Z,et al.Efficient generation of Myostatin mutations in pigs using the CRISPR/Cas9 system[J].Scientific Reports,2015,5:16623.
[11] GU C S,LIU L Q,XU C,et al.Reference gene selection for quantitative real-time RT-PCR normalization in iris.Lactea var.chinensis roots under cadmium,lead,and salt stress conditions[J].The Scientific World Journal,2014,2014(2):1-7.
[12] 张蓉,施晓丽,陈朝桂,等.实时荧光定量PCR研究蛋鸡组织内参基因表达的筛选[J].中国畜牧兽医,2017,44(8):2226-2233. ZHANG R,SHI X L,CHEN C G,et al.Screening of tissue reference gene expression in laying hens by real-time fluorescence quantitative PCR[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2226-2233.(in Chinese)
[13] 彭馥芝,冉茂良,翁波,等.猪睾丸组织定量PCR分析中内参基因的选择[J].中国农业科学,2017,50(15):3033-3041. PENG F Z,RAN M L,WENG B,et al.Selection of reference genes in quantitative PCR analysis of pig testis tissue[J].Scientia Agricultura Sinica,2017,50(15):3033-3041.(in Chinese)
[14] 王轶敏,代阳,刘新峰,等.牛骨骼肌卫星细胞的分离鉴定和诱导分化[J].中国畜牧兽医,2014,41(7):142-147. WANG Y M,DAI Y,LIU X F,et al.Isolation identification and induction differentiation of bovine skeletal muscle satellite cells[J].Chinese Animal Husbandry & Veterinary Medicine,2014,41(7):142-147.(in Chinese)
[15] 代阳,王轶敏,刘新峰,等.兔骨骼肌卫星细胞成肌诱导分化及鉴定[J].黑龙江畜牧兽医,2014(21):176-179. DAI Y,WANG Y M,LIU X F,et al.Myogenic differentiation and identification of rabbit skeletal muscle satellite cells[J].Heilongjiang Animal Science and Veterinary Medicine,2014(21):176-179.(in Chinese)
[16] 丁向彬,张蔚然,张俊星,等.lncRNA-HZ5对牛骨骼肌卫星细胞成肌分化的调控作用研究[J].天津农学院学报,2017,24(3):64-68. DING X B,ZHANG W R,ZHANG J X,et al.Regulation of lncRNA-HZ5 on myogenic differentiation of bovine skeletal muscle satellite cells[J].Journal of Tianjin Agricultural University,2017,24(3):64-68.(in Chinese)
[17] 孙海成,吕晓楠,佟广香,等.尖裸鲤实时荧光定量PCR内参基因的筛选[J].大连海洋大学学报,2019,34(3):370-375. SUN H C,LYU X N,TONG G X,et al.Screening of internal reference genes in Real-time fluorescence quantitative PCR of naked carp[J].Journal of Dalian Fisheries University,2019,34(3):370-375.(in Chinese)
[18] 唐培安,张棋麟,薛昊,等.印度谷螟实时荧光定量PCR内参基因的选择[J].中国科学,2016,46(10):1201-1209. TANG P A,ZHANG Q L,XUE H,et al.Reference genes in quantitative real-time PCR of Plodia interpunctella(Lepidoptera:Pyralidate)[J].Scientia Sinica (Vitae),2016,46(10):1201-1209.(in Chinese)
[19] 齐波,朱荣生,王怀中,等.测定猪不同组织基因表达时RT-PCR内参基因的选择[J].家畜生态学报,2017,38(6):8-12. QI B,ZHU R S,WANG H Z,et al.Selection of RT-PCR reference genes for gene expression in different tissues of pigs[J].Journal of Domestic Animal Ecology,2017,38(6):8-12.(in Chinese)
[20] 马红,刘宇,汪亮,等.民猪外周血单核细胞实时荧光定量PCR内参基因的筛选[J].中国畜牧兽医,2017,44(12):3426-3433. MA H,LIU Y,WANG L,et al.Screening of the internal reference genes of real-time fluorescent quantitative PCR for peripheral blood mononuclear cells of Minzhu[J].China Animal Husbandry & Veterinary,2017,44(12):3426-3433.(in Chinese)
[21] 刘方舟.稻飞虱翅发育基因对翅型分化的调控作用研究[D].武汉:华中农业大学,2018. LIU F Z.Study on the regulation of wing development gene of rice planthopper on wing type differentiation[D].Wuhan:Huazhong Agricultural University,2018.(in Chinese)
[22] 刘瑞凿,刘真,吴明明,等.绵羊胚胎发育中后期骨骼肌BTG2/3的表达动态及其与Myostatin的关系[J].畜牧兽医学报,2015,46(11):1916-1923. LIU R Z,LIU Z,WU M M,et al.The expression of BTG2/3 in skeletal muscle and its relationship with Myostatin during the middle and late embryonic development of sheep[J].Chinese Journal of Animal and Veterinary Sciences,2015,46(11):1916-1923.(in Chinese)
[23] 王红娜,孙洪新,张英杰,等.干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响[J].畜牧兽医学报,2018,49(1):46-54. WANG H N,SUN H X,ZHANG Y J,et al.Effect of interfering MSTN on proliferation and differentiation of sheep myoblasts and expression of related genes[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(1):46-54.(in Chinese)
[24] 卢靖坤.MSTN蛋白结构变异对牛肌肉生长的影响[D].呼和浩特:内蒙古大学,2018. LU J K.Effect of structural variation of MSTN protein on beef muscle growth[D].Hohhot:Inner Mongolia University,2018.(in Chinese)
[25] 吴建阳,何冰,杜玉洁,等.利用geNorm、NormFinder和BestKeeper软件进行内参基因稳定性分析的方法[J].现代农业科技,2017,5:278-281. WU J Y,HE B,DU Y J,et al.A method for stability analysis of internal reference genes using geNorm,NormFinder and BestKeeper software[J].Modern Agricultural Science and Technology,2017,5:278-281.(in Chinese)