巴马小型猪组织qRT-PCR内参基因的筛选  

Screening of Reference Genes for qRT-PCR in Bama Minipig(Sus scrofa)Tissues

作　　者：陈楚杰 裴杨莉 刘璐璐 杨亚岚 张垒霞 李华 李奎 CHEN Chu-Jie;PEI Yang-Li;LIU Lu-Lu;YANG Ya-Lan;ZHANG Lei-Xia;LI Hua;LI Kui(Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding/School of Life Sciences and Engineering,Foshan University,Foshan 528225,China;Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]广东省动物分子设计与精准育种重点实验室/佛山科学技术学院生命科学与工程学院,佛山528225 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2020年第6期1105-1113,共9页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(2018ZX08010-08B);国家自然科学青年基金(31702088);广东省动物分子设计与精准育种重点实验室开放课题(2018A07);佛山科学技术学院高层次人才科研启动项目(2018)。

摘　　要：qRT-PCR是检测细胞、组织基因表达的重要方法之一。而选择合适的内参基因是准确计算目标基因表达水平并进行基因功能研究的关键。本研究使用qRT-PCR技术检测了9个候选基因:拓扑异构酶-2β基因(topoisomerase(DNA)Ⅱβ,Top2b)、β-肌动蛋白基因(β-actin,Actb)、核糖体蛋白S18基因(ribosomal protein S18,Rps18)、肽基脯氨酰异构酶A基因(peptidylprolyl isomerase A,Ppia)、18S核糖体RNA基因(18S ribosomal RNA,18S)、TATA盒结合蛋白基因(TATA-box binding protein,Tbp)、羟甲基胆素合成酶基因(hydroxymethylbilane synthase,Hmbs)、核糖体蛋白L4基因(ribosomal protein L4,Rpl4)和β-2-微球蛋白基因(β-2-microglobulin,B2m),在15日龄巴马小型猪(Sus scrofa)的心、肝、脾、肺、肾、肌肉和皮下脂肪7个组织上的表达情况,并通过geNorm、NormFinder和BestKeeper程序对结果进行分析。基因表达稳定性结果表明,Rps18、Rpl4、Ppia和Tbp在所有组织中稳定性最高。而单个组织内只需使用两个内参基因即可标准化目标基因的表达,且不同组织的最优内参基因组合不同。本结果为选择巴马小型猪内参基因提供参考,进而为巴马小型猪的肉质、产仔数、发育、繁殖和疾病等相关基因的功能研究提供理论基础。qRT-PCR is one of the important methods for investigating mRNA expression levels in the cells and tissues.However,the selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis.This study examined the expression of 9 internal genes(topoisomerase(DNA)Ⅱβ(Top2b),β-actin(Actb),ribosomal protein S18(Rps18),peptidylprolyl isomerase A(Ppia),18S ribosomal RNA(18S),TATA-box binding protein(Tbp),hydroxymethylbilane synthase(Hmbs),ribosomal protein L4(Rpl4)andβ-2-microglobulin(B2m)for heart,liver,spleen,lung,kidney,muscle,subcutaneous fat from three 15 d Bama minipigs(Sus scrofa)using qRT-PCR technique,and 3 well-known software packages:geNorm,NormFinder and BestKeeper were used to analyze the results.Rps18,Rpl4,Ppia and Tbp were found to own the highest stability across tissues.Only 2 internal reference genes could standardize the expression of target genes in the same tissue.And in the different tissue,the optimal gene combination was different.The results of this study provide a reference information on Bama minipig to select the most suitable internal reference gene,and it could provide a theoretical basis for the research of gene function about meat quality,litter size,development,reproduction,disease and so on of Bama minipig.

关 键 词：QRT-PCR 基因表达 内参基因 巴马小型猪 

分 类 号：S828[农业科学—畜牧学]