山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析

doi:10.11843/j.issn.0366-6964.2018.05.005

摘要/Abstract

摘要：

旨在通过比较9个内参基因在山羊肌内前体脂肪细胞诱导分化不同时期的表达水平进而最终确定适合山羊肌内前体脂肪细胞分化调控研究最佳的内参基因。本研究利用胶原酶消化法获得山羊肌内前体脂肪细胞，并以分别诱导0、1、2、3、5和6 d的细胞为研究对象，利用荧光定量PCR （Real-time quantitative PCR，qPCR）技术和geNorm、NormFinder和BestKeeper程序来检测和分析9个候选内参基因（GAPDH、PPIA、18S rRNA、PPIB、UXT、RPLP0、ACTB、EIF3K和TBP）表达的稳定性，并以KLF3和KLF12的实际表达水平进行校正分析。geNorm和NormFinder程序分析表明，UXT稳定性相对最好；而BestKeeper程序分析发现，PPIB表达相对最平稳；3种软件分析结果均表明，同时使用UXT和PPIB可以准确校正在山羊肌内前体脂肪细胞诱导分化不同时期目标基因的表达；通过对KLF3和KLF12表达量的分析发现，选用筛选结果中最稳定（UXT和PPIB、UXT）和最不稳定（EIF3K和18S rRNA）的内参基因进行归一化分析结果有差异。在山羊肌内前体脂肪细胞诱导分化过程中，UXT基因是适合于山羊肌内前体脂肪细胞的成脂诱导分化研究最稳定有效的内参基因，同时使用UXT和PPIB作为内参基因能够获得更加精确的目标基因表达结果，这为后续山羊前体脂肪细胞分化调控相关基因的功能研究奠定了基础。

关键词: 山羊, 肌内前体脂肪细胞, 内参基因, 实时荧光定量PCR

Abstract:

The aim of this study was to determine the most suitable reference genes in different periods of goat intramuscular preadipocytes differentiation in goat by comparing the expression levels of 9 candidate reference genes. The intramuscular preadipocytes of goat were obtained by collagenase digestion technique. The adipocytes induced for 0, 1, 2, 3, 5 and 6 d were used in this study. The expression levels of the 9 candidate reference genes (GAPDH, PPIA, 18S rRNA, PPIB, UXT, RPLP0, ACTB, EIF3K and TBP) were detected by real-time quantitative PCR (qPCR), and the expression stability was evaluated by geNorm, NormFinder and BestKeeper programs, individually. Meanwhile, the suitability of the selected reference genes for normalization was further assessed by the expression of KLF3 and KLF12. The results showed that UXT was identified as the most suitable reference gene by both geNorm and NormFinder analysis, while the PPIB was identified as the most suitable reference gene by BestKeeper analysis. The combined use of UXT and PPIB as the reference genes resulted in the optimal selection for normalization of analyzed genes in different periods of intramuscular preadipocytes differentiation in goat based on the analysis of geNorm, NormFinder and BestKeeper; Furthermore, the expression levels of KLF3 and KLF12 normalized by the most stable reference genes (UXT and PPIB, UXT) were significantly different from that normalized by the least stable ones (EIF3K, 18S rRNA). In conclusion, the expression of UXT was the most stable and it was the most suitable reference genes during the differentiation of goat intramuscular preadipocyte in goat, meanwhile the combined use of UXT and PPIB was found to lead to a more accurate result for target genes normalization. These results provided a foundation for functional research of genes related to preadipocytes differentiation in goats.

Key words: goat, intramuscular preadipocytes, reference gene, real-time quantitative PCR

中图分类号:

S827.2

许晴, 林森, 朱江江, 王永, 林亚秋. 山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J]. 畜牧兽医学报, 2018, 49(5): 907-918.

XU Qing, LIN Sen, ZHU Jiang-jiang, WANG Yong, LIN Ya-qiu. The Expression Stability Analysis of Reference Genes in the Process of Goat Intramuscular Preadipocytes Differentiation in Goat[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(5): 907-918.

参考文献

[1] WOOD J D,ENSER M,FISHER A V,et al.Fat deposition,fatty acid composition and meat quality:A review[J].Meat Sci,2008,78(4):343-358.
[2] BONNET M,BERNARD L,BES S,et al.Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,2013,7(8):1344-1353.
[3] SUGDEN K,PARIANTE C M,MCGUFFIN P,et al.Housekeeping gene expression is affected by antidepressant treatment in a mouse fibroblast cell line[J].J Psychopharmacol,2010,24(8):1253-1259.
[4] RADONIC A,THULKE S,MACKAY I M,et al.Guideline to reference gene selection for quantitative real-time PCR[J].Biochem Biophys Res Commun,2004,313(4):856-862.
[5] 陈利,赵薇,占思远,等.山羊不同组织及不同发育时期骨骼肌内参基因的表达稳定性分析[J].畜牧兽医学报,2014,45(8):1228-1236.
CHEN L,ZHAO W,ZHAN S Y,et al.The expression stability analysis of reference genes in the different tissues and skeletal muscle of different development periods in goat[J].Acta Veterinaria et Zootechnica Sinica,2014,45(8):1228-1236.(in Chinese)
[6] BAI W L,YIN R H,ZHAO S J,et al.Technical note:Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle[J].J Dairy Sci,2014,97(2):902-910.
[7] ZHANG Y,ZHANG X D,LIU X,et al.Reference gene screening for analyzing gene expression across goat tissue[J].Asian-Australas J Anim Sci,2013,26(12):1665-1671.
[8] 张晓东.山羊卵巢microRNA的鉴定及生物学功能分析[D].合肥:安徽农业大学,2013.
ZHANG X D.Identification and biological role analysis of microRNA in goat ovaries[D].Hefei:Anhiu Agricultural University,2013.(in Chinese)
[9] VORACHEK W R,HUGEJILETU,BOBE G,et al.Reference gene selection for quantitative PCR studies in sheep neutrophils[J].Int J Mol Sci,2013,14(6):11484-11495.
[10] 马志远,翁秀秀,李飞,等.湖羊小肠黏膜内参基因筛选[J].动物营养学报,2015,27(11):3478-3484.
MA Z Y,WENG X X,LI F,et al.Selection of reference genes in small intestinal mucosa of Hu Lambs[J].Chinese Journal of Animal Nutrition,2015,27(11):3478-3484.(in Chinese)
[11] JEDRZEJCZAK M,SZATKOWSKA I.Bovine mammary epithelial cell cultures for the study of mammary gland functions[J].in vitro Cell Dev Biol Anim,2014,50(5):389-398.
[12] CHOUDHARY R,KUMAR S,SINGH S V,et al.Validation of putative reference genes for gene expression studies in heat stressed and α-MSH treated melanocyte cells of Bos indicus using real-time quantitative PCR[J].Mol Cell Probes,2016,30(3):161-167.
[13] GUO Y,PENNELL M L,PEARL D K,et al.The choice of reference gene affects statistical efficiency in quantitative PCR data analysis[J].Biotechniques,2013,55(4):207-209.
[14] 李倩,林亚秋,朱江江,等.山羊FGF 21基因克隆及其在肌内脂肪细胞中的表达模式研究[J].畜牧兽医学报,2017,48(1):31-38.
LI Q,LIN Y Q,ZHU J J,et al.Cloning of goat FGF 21 gene and its expression pattern in intramuscular adipocyte[J].Acta Veterinaria et Zootechnica Sinica,2017,48(1):31-38. (in Chinese)
[15] VANDESOMPELE J,DE P K,PATTYN F,et al.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biol,2002,3(7):research0034.
[16] ANDERSEN C L,JENSEN J L,ØRNTODT T F.Normalization of real-time quantitative reverse transcription PCR data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets[J].Cancer Res,2004,64(15):5245-5250.
[17] PFAFFL M W,TICHOPAD A,PRGOMET C,et al.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:Bestkeeper-Excel-based tool using pair-wise correlations[J].Biotechnol Lett,2004,26(6):509-515.
[18] ZHU W Z,LIN Y Q,LIAO H H,et al.Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle[J].PLoS One,2015,10(3):e0121280.
[19] KLENKE S,RENCKHOFF K,ENGLER A,et al.Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments[J].Naunyn Schmiedeberg's Arch Pharmacol,2016,389(12):1353-1366.
[20] 张蓉,施晓丽,陈朝桂,等.实时荧光定量PCR研究蛋鸡组织基因表达的内参基因筛选[J].中国畜牧兽医,2017,44(8):2226-2233.
ZHANG R,SHI X L,CHEN C G,et al.Screening of reference genes for gene expression in layer hens tissues using real-time quantitative PCR[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2226-2233.(in Chinese)
[21] 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 reference genes of real-time quantitative PCRin the gene expression of different tissues in pig[J].Acta Ecologiae Animalis Domastici,2017,38(6):8-12.(in Chinese)
[22] 李迪,吴萍,何美凤,等.qRT-PCR分析鳜鱼内参基因的筛选[J].生命科学研究,2016,20(3):214-217.
LI D,WU P,HE M F,et al.Screening of reference genes in siniperca chuatsi for qRT-PCR analysis[J].Life Science Research,2016,20(3):214-217.(in Chinese)
[23] GARCIA-CRESPO D,JUSTE R A,HURTADO A.Selection of ovine housekeeping genes for normalisation by real-time RT-PCR;analysis of PrP gene expression and genetic susceptibility to scrapie[J].BMC Vet Res,2005,1:3.
[24] ZHAO H W,WANG Q,ZHANG H T,et al.UXT is a novel centrosomal protein essential for cell viability[J].Mol Biol Cell,2005,16(12):5857-5865.
[25] 秦彤,王皓宇,郝海生,等.日粮精粗比对围产期奶牛乳腺内参基因表达的影响[J].华北农学报,2013,28(4):75-78.
QIN T,WANG H Y,HAO H S,et al.Reference gene expression in mammary tissue of periparturient cows is affected by dietary forage to concentrate ratio[J].Acta Agriculture Boreali-Sinica,2013,28(4):75-78.(in Chinese)
[26] BIONAZ M,LOOR J J.Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle[J].Physiol Genomics,2007,29(3):312-319.
[27] MUHAGHEGH-DOLATABADY M,HOSSAINY-DOLATABAD H H,ARIJLO E H,et al.Validation of reference genes for real time PCR normalization in milk somatic cells of holstein dairy cattle[J].Iran J Appl Anim Sci,2017,7(2):229-234.
[28] 王慧,付金栋,刘京营,等.亲环素B、D在肝纤维化模型中的变化及其意义[J].中华肝脏病杂志,2012,20(9):705-706.
WANG H,FU J D,LIU J X,et al.Study of Cyclophilin B and D in rat liver fibrosis models[J].Chinese Journal of Hepatology,2012,20(9):705-706. (in Chinese)
[29] 乐飞,李健文,冯波,等.PPIB蛋白对结肠癌成瘤的影响和低氧机制[J].外科理论与实践,2015,20(6):505-509.
LE F,LI J W,FENG B,et al.Effect of PPIB on tumorigenesis in colon cancer and hypoxia-mediated mechanism[J].Journal of Surgery Concepts & Practice,2015,20(6):505-509. (in Chinese)
[30] PACHOT A,BLOND J L,MOUGIN B,et al.Peptidylpropyl isomerase B (PPIB):a suitable reference gene for mRNA quantification in peripheral whole blood[J].J Biotechnol,2004,114(1-2):121-124.
[31] 陈瑞,杨晓农,曾婉秋,等.家兔不同发育阶段和组织中内参基因的稳定性分析[J].畜牧兽医学报,2016,47(3):477-483.
CHEN R,YANG X N,ZENG W Q,et al.Expression stability analysis of reference genes in different development periods and tissues in oryctolagus cuniculus[J].Acta Veterinaria et Zootechnica Sinica,2016,47(3):477-483.(in Chinese)