不同产羔数辽宁绒山羊各组织内参基因的筛选  

Screening of reference genes in different tissues of Liaoning cashmere goats with different lambing numbers

作　　者：薛丽娜 毛杨毅[1] 罗惠娣[1] 赵鹏[1] 李俊 郭慧慧 张丽[1] 武守艳 XUE Lina;MAO Yangyi;LUO Huidi;ZHAO Peng;LI Jun;GUO Huihui;ZHANG Li;WU Shouyan(College of Animal Science,Shanxi Agricultural University,Jinzhong 030801,China;College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030801,China)

机构地区：[1]山西农业大学动物科学学院,山西晋中030801 [2]山西农业大学动物医学学院,山西晋中030801

出　　处：《黑龙江畜牧兽医》2022年第11期1-6,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家现代农业产业技术体系项目(CARS-39-24);山西省科技攻关项目(20120311024-1);山西省科技创新团队项目(201705D131028-20);山西省农业产业发展科技引领工程项目(2019CYYL-12);山西省重点研发计划项目(201903D221003)。

摘　　要：为了筛选不同产羔数辽宁绒山羊母羊各组织中稳定表达的内参基因,试验随机选取同期发情后的4岁产单羔和双羔母羊各3只,采集心脏、肝脏、脾脏、肺脏、肾脏、肌肉、下丘脑、垂体、子宫角、卵巢,取各器官部分组织提取总RNA,检测总RNA浓度和纯度后合成cDNA,设计U6、ACTβ、B2M、18S rRNA、GAPDH、RPLPO内参基因引物,检测各引物的特异性和扩增效率,采用实时荧光定量PCR方法检测6个常见内参基因在以上组织中的表达情况,通过geNorm、NormFinder、BestKeeper和RefFin-der软件综合筛选出稳定表达的内参基因,并用geNorm软件分析最佳内参基因个数。结果表明:提取到的总RNA浓度和纯度较高,设计的引物特异性和扩增效率(96.53%~107.23%)较好,各内参基因在不同产羔数辽宁绒山羊母羊全部组织中均大量表达,且表达水平存在差异,Ct值在8.18~17.61之间。经geNorm、NormFinder、BestKeeper软件筛选发现不同产羔数辽宁绒山羊母羊性腺轴组织和全部组织中最稳定的内参基因不同,可能是软件算法不同造成的;经geNorm软件分析,最佳内参基因个数均为2个。经RefFinder软件分析在下丘脑中最稳定的内参基因组合是RPLPO和B2M基因,垂体中最稳定的内参基因组合是RPLPO和U6基因,卵巢中最稳定的内参基因组合是RPLPO和GAPDH基因,子宫角中最稳定的内参基因组合是GAPDH和18S rRNA基因。说明在不同产羔数辽宁绒山羊母羊全部组织中及性腺轴下丘脑、垂体、卵巢、子宫角各组织中最稳定的内参基因的组合分别为是U6和RPLPO,RPLPO和B2M,RPLPO和U6,RPLPO和GAPDH,GAPDH和18S rRNA。In order to screen the reference genes stably expressed in tissues of Liaoning cashmere goat ewes with different lambing numbers, in this experiment, 3 ewes with single lambing and 3 ewes with double lambing at 4 years old after estrus were randomly selected. Their, liver, spleen, lung, kidney, muscle, hypothalamus, pituitary, uterine horn and ovary were collected to extract total RNA from partial tissues of each organ. After detecting the concentration and purity of total RNA, cDNA was synthesized, and reference primers for U6, ACTβ, B2 M, 18 S rRNA, GAPDH and RPLPO were designed to detect the specificity and amplification efficiency of each primer. The expression of six candidate reference genes in the tissues mentioned above was detected by real-time fluorescence quantitative PCR. GeNorm, NormFinder, BestKeeper and RefFinder software were combined to screen out the reference genes with stable expression, and geNorm software was used to analyze the optimal number of reference genes. The results showed that the extracted total RNA concentration and purity were high, and the designed primers had good specificity and amplification efficiency(96.53%-107.23%). All reference genes were largely expressed in all tissues of Liaoning cashmere goats with different lambing numbers, and the expression levels were different, and the Ct value was between 8.18-17.61. Through the screening of geNorm, NormFinder, BestKeeper and RefFinder software, it was found that the most stable reference genes in gonadal axis tissues and all tissues of different of lambing numbers of Liaoning cashmere goats were different, which might be caused by different software algorithms. According to the analysis of geNorm software, the best number of reference genes was 2. According to the analysis of RefFinder software, RPLPO and B2 M were the most stable combination of reference genes in hypothalamus;RPLPO and U6 were the most stable combination of reference genes in pituitary;RPLPO and GAPDH were the most stable combination of reference genes in o

关 键 词：不同产羔数 辽宁绒山羊母羊 性腺轴 表达量 内参基因筛选 

分 类 号：S827[农业科学—畜牧学] Q78[农业科学—畜牧兽医]