金堂黑山羊不同组织内参基因筛选与稳定性分析  

Screening of suitable reference genes for RT-qPCR normalization in the different tissues of Jintang Black Goat

作　　者：张楠驰 李娟[1] 王利[1] 魏勇[2] ZHANG Nan-chi;LI Juan;WANG Li;WEI Yong(Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Ministry of Education and Sichuan Province,Southwest Minzu University,Chengdu 610041,China;Animal Genetics and Breeding Key Laboratory of Sichuan Province,Animal Science Academy of Sichuan Province,Chengdu 610066,China;A Ba Vacational College,A Ba 623200,China)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部和四川省重点实验室,四川成都610041 [2]四川省畜牧科学研究院动物遗传育种四川省重点实验室,四川成都610066 [3]阿坝职业学院,四川阿坝623200

出　　处：《中国预防兽医学报》2020年第11期1109-1115,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：四川省肉羊创新团队防疫岗位(sccxtd-2020-14);农业农村部农业重大技术协同推广计划试点四川省肉羊高效生产配套技术推广应用。

摘　　要：为筛选检测金堂黑山羊不同组织转录水平的荧光定量PCR方法中的最佳内参基因,本实验首先利用荧光定量PCR(qPCR)检测16个候选基因在金堂黑山羊心、肝、脾、瘤胃、背最长肌和臀肌中的转录水平,并利用geNorm、NormFinder、BestKeeper程序分析候选基因转录水平的稳定性。再以最佳内参基因组合、最佳内参基因和2个转录水平稳定性相对最差的候选基因作为内参基因,经qPCR检测黑山羊心、脾和臀肌中相容性复合体Ⅱ类基因(MHC class II DQ-beta 1 chain gene,DQB1)的转录水平,验证筛选基因的稳定性。结果显示,不同候选基因在不同组织中的转录水平稳定性存在一定的差异性。经geNorm、NormFinder、BestKeeper程序分析得出最稳定内参基因为PPIB和YWHAZ,相对最不稳定内参基因为18S rRNA、TOP2β和EIF3K。以最佳内参基因组合(PPIB和YWHAZ)、最佳内参基因(PPIB)和两个最不稳定基因(TOP2β和EIF3K)作为内参基因检测DQB1在黑山羊心、脾和臀肌中转录水平,前两者分别与后两者所得到的DQB1转录水平差异显著(p<0.05),验证了本实验所筛选内参基因的稳定性。本实验首次筛选出了金堂黑山羊最佳内参基因组合为PPIB和YWHAZ,为金堂黑山羊不同组织qPCR分析中的内参基因选择提供参考。The aim of this study was to determine the most suitable reference genes in different tissues(heart,liver,spleen,rumen,longissimus dorsi and gluteus)of Jingtang black goat by comparing the transcription levels of 16 candidate genes.Sixteen candidate housekeeping genes in six tissues of Jintang black goat were assessed using RT-qPCR and three algorithms,including geNorm,Norm Finder and Bestkeeper.Then appropriate reference genes were validated by MHC class II DQ-beta 1 chain gene(DQB1).The results showed there are differentces in the stability of transcription level of different candidate housekeeping genes in different tissues.PPIB and YWHAZ were the most stable genes,where as 18S rRNA,TOP2βand EIF3K were the least stable genes in Jintang black goat's six tissues.The combination of PPIB and YWHAZ were the optimal reference genes for gene transcription level analysis by RT-qPCR in Jingtang black goat's six tissues.The transcription levels of the normalized DQB1 by the combination of most stable reference genes(PPIB and YWHAZ)and most stable reference genes(PPIB)were significantly different(p<0.05)from that normalized by the least stable ones(TOP2β,EIF3K),confirming the stability of the selected internal reference genes.This study showed that PPIB and YWHAZ were the most stable reference genes in Jintang black goat's six tissues,providing a pair of reference genes to analyze mRNA transcription levels in Jintang black goat tissues.

关 键 词：金堂黑山羊 内参基因 荧光定量PCR 

分 类 号：S827[农业科学—畜牧学]