山羊不同组织实时荧光定量PCR内参基因的筛选

徐颂为; 黄忍; 杨璐; 向华; 张焕容

doi:10.13881/j.cnki.hljxmsy.2021.02.0220

山羊不同组织实时荧光定量PCR内参基因的筛选

西南民族大学畜牧兽医学院

基金项目:

西南民族大学研究生创新型科研项目(CX2020SZ52)

详细信息

作者简介:
徐颂为(1995—),女,硕士研究生,研究方向为动物传染病学,156952592@qq.com

通讯作者:
向华(1986—),女,助理研究员,博士,研究方向为畜禽传染病学,xianghua2008411@163.com

张焕容(1968—),女,教授,博士,研究方向为动物传染病快速诊断与防治技术,727746768@qq.com

中图分类号: S827
计量
- 文章访问数: 0
- HTML全文浏览量: 0
- PDF下载量: 0
出版历程
- 收稿日期: 2021-02-22
- 刊出日期: 2021-12-19

Screening of reference genes for real-time fluorescence quantification PCR in different goat tissues

College of Animal Science and Veternary Medicine, Southwest Minzu University

摘要

摘要: 为了筛选出在山羊组织中能稳定表达的内参基因,试验采集1月龄简州大耳羊的心脏、肝脏、脾脏、肺脏、肾脏、肠、皮肤、肌肉、淋巴结和唇10个组织样品,选取甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)、组蛋白3.3(H3-histone family 3A, H3F3A)、beta-肌动蛋白(actin-beta, ACTB)和肽基脯氨酰异构酶(peptidylprolyl isomerase, PPIA)4个基因做为候选内参基因,采用实时荧光定量PCR(RT-qPCR)技术检测上述10个组织样品的中4个候选内参基因的表达情况,利用geNorm软件分析4个候选基因的定量数据,筛选出在山羊组织中表达稳定的内参基因。结果表明:成功克隆GAPDH、H3F3A、ACTB和PPIA 4个内参基因,4个内参基因的RT-qPCR扩增曲线正常,无引物二聚体和非特异性条带产生,PCR产物单一,绘制的标准曲线上各点在同一条直线上。4个内参基因的稳定度排序为H3F3A>PPIA>ACTB>GAPDH,相对表达量依次为PPIA>H3F3A>ACTB>GAPDH。说明PPIA和H3F3A基因表达水平较高,稳定性较好,可作为后续研究山羊组织中功能基因表达量的最佳内参基因。
- 山羊 /
- 内参基因 /
- 实时荧光定量PCR /
- geNorm软件 /
- 稳定性
Abstract: In order to screen out reference genes that could be stably expressed in goat tissue, ten tissues of heart, liver, spleen, lung, kidney, intestine, skin, muscle, lymph and lip of one-month-old Jianzhou big ear sheep were collected in the experiment. Four genes, glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, H3-histone family 3 A（H3 F3 A）, actin-beta（ACTB） and peptidylprolyl isomerase（PPIA）, were selected as candidate reference genes. Real-time fluorescence quantification PCR（RT-qPCR） was used to detect the expression of four candidate reference genes in the above ten tissue samples, and the quantitative data of four candidate genes were analyzed using the geNorm program to select reference genes with stable expression in goat tissues. The results showed that four reference genes（including GAPDH, H3 F3 A, ACTB and PPIA） were successfully cloned. The RT-qPCR amplification curves of the four reference genes were normal, without primer dimer and non-specific bands were produced, the PCR products were single, and each point on the drawn standard curve was on the same line. The stability of the four reference genes was H3 F3 A ＞ PPIA ＞ ACTB ＞ GAPDH, and the relative expression was PPIA ＞ H3 F3 A ＞ ACTB ＞ GAPDH. It indicated that PPIA and H3 F3 A genes had high expression level and good stability, and could be used as the best reference genes for subsequent study of the expression of functional genes in goat tissues.
- goat /
- reference genes /
- real-time fluorescence quantification PCR /
- geNorm software /
- stability

HTML全文

参考文献(26)

[1]	GIULIETTI A,OVERBERGH L,VALCKX D,et al.An overview of real-time quantitative PCR:Applications to quantify cytokine gene expression[J].Methods,2001,25(4):386-401.
[2]	FLEIGE S,PFAFFL M W.RNA integrity and the effect on the real-time qRT-PCR performance[J].Mol Aspects Med,2006,27(2/3):126-139.
[3]	台玉磊,韩立强,杨国庆,等.仔猪组织基因表达中实时定量PCR内参基因的选择[J].农业生物技术学报,2010,18(04):732-736.
[4]	JUNG M,RAMANKULOV A,ROIGAS J,et al.In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR[J].BMC Mol Biol,2007,8:47.
[5]	张艳君,朱志峰,陆融,等.基因表达转录分析中内参基因的选择[J].生物化学与生物物理进展,2007,34(05):546-550.
[6]	贺淹才.肌动蛋白和肌动蛋白基因的研究进展[J].生命的化学,2002,22(03):248-250.
[7]	付国良,黄晓红.甘油醛-3-磷酸脱氢酶功能的研究进展[J].生物物理学报,2013,29(03):181-191.
[8]	许晴,林森,朱江江,等.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(05):907-918.
[9]	陈修文,汤显斌,王铁延,等.骨巨细胞瘤中H3F3A基因突变检测及其临床意义[J].临床与实验病理学杂志,2019,35(09):1115-1116,1118.
[10]	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 (05):278-281.
[11]	孙金梅.山羊的起源和进化[J].中国养羊,1997 (01):7-9,20.
[12]	贾青,常洪,马章全.山羊的起源驯化和品种形成[J].河北农业大学学报,1997,20(02):68-71.
[13]	PUECH C,DEDIEU L,CHANTAL I,et al.Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle,sheep and goats[J].BMC Vet Res,2015,11:65.
[14]	陶蓉,李慧,孙宇航,等.美国白蛾内参基因的鉴定及筛选[J].林业科学,2019,55(09):111-120.
[15]	RADONIC′A,THULKE S,MACKAY L M,et al.Guideline to reference gene selection for quantitative real-time PCR[J].Biochem Biophys Res Commun,2004,313(4):856-862.
[16]	XIE F,SUN G,STILLER J W,et al.Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database[J].PLoS One,2011,6(11):e26980.
[17]	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):467-470.
[18]	MAMO S,GAL A B,BODO S,et al.Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro[J].BMC Dev Biol,2007,7:14.
[19]	OHL F,JUNG M,XU C,et al.Gene expression studies in prostate cancer tissue:which reference gene should be selected for normalization[J].J Mol Med (Berl),2005,83(12):1014-1024.
[20]	ZHANG X,DING L,SANDFORD A J.Selection of reference genes for gene expression studies in human neutrophils by real-time PCR[J].BMC Mol Biol,2005,6:4.
[21]	LI L,JIANG J,WANG L,et al.Expression patterns of peroxisome proliferator-activated receptor gamma 1 versus gamma 2,and their association with intramuscular fat in goat tissues[J].Gene,2013,528(2):195-200.
[22]	ZHU H,PARK S,SCHEFFLER J M,et al.Porcine satellite cells are restricted to a phenotype resembling their muscle origin[J].JAnim Sci,2013,91(10):4684-4691.
[23]	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.
[24]	王子东,苏建国,段彪,等.牛转FAD3B基因胎儿成纤维细胞中内参基因的稳定性评估[J].生物技术通报,2012,11:118-126.
[25]	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.
[26]	朱鑫,吴巧,冯晓辉,等.H1N1猪流感病毒感染猪血管内皮细胞后内参基因的筛选[J].西南民族大学报,2014,40 (4):490-494.

施引文献(3)

期刊类型引用(2)

1.	霍泱安，李小雪，孙郴，李齐发，杜星. 猪卵泡发育过程中颗粒细胞内参基因表达稳定性分析. 南京农业大学学报. 2024(04): 710-720 . 百度学术
2.	吴昊天，张振兴，陈思，刘志勇，安琪，陈杰，程逸文，刘昂，何美荣，蒋俊明，陈巧玲，王凤阳，杜丽，满初日嘎. 布鲁氏菌脂多糖刺激山羊支气管上皮细胞的全转录组分析. 基因组学与应用生物学. 2022(Z1): 1851-1861 . 百度学术