内参基因表达稳定性的高低将决定着实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)分析结果的可靠程度.本实验旨在筛选不同浓度氨基葡萄糖(glucosamine,GLuN)处理下产蛋后期笼养蛋鸡(Gallus gallus domesticus)中稳定可靠的内参基因,以确保产蛋后期蛋鸡基因表达分析结果的可靠性.实验选用500日龄产蛋后期海兰灰蛋鸡作为研究对象,对照组饲喂玉米-豆粕型基础饲粮,实验组分别在基础饲粮中添加0.4％、0.6％和0.8％ GLuN,运用qRT-PCR技术对核糖体蛋白S2(ribosomal protein S2,RPS2)、肌动蛋白(β-Actin)、3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、羟甲基胆色烷合成酶(hydroxymethyl biliary synthetase,HMBS)、端粒结合蛋白(TATA-box binding protein,TBP)、次黄嘌呤鸟嘌呤磷酸核糖转移酶1(hypoxanthine phosphoribosyltransferase 1,HPRT1)、微管蛋白(tubulin beta class,TUBB)、琥珀酸脱氢酶复合物亚单位A(succinate dehydrogenase complex flavoprotein subunit A,SDHA)、核糖体蛋白L4 (ribosomal protein L4,RPL4)和微球蛋白(beta-2 microglobulin,B2M等10个内参基因在不同组织中(心、肝、肺、肾、十二指肠、胫骨、胰和子宫)的表达情况进行分析,同时借助循环阈值法(cycle threshold,Cq)、Delta循环阈值法(Delta cycle threshold,Delta CT)和geNorm、NormFinder、RefFinder软件对每个内参基因的表达稳定性进行评估.结果显示,在利用原始Cq平均值分析时发现,同一内参基因在不同处理和不同组织中的表达量不同,说明所选择的10个候选内参基因在组织间存在一定的表达差异性;Cq值法分析显示,RPS2基因的表达稳定性最好;Delta CT法分析发现,不同的GLuN水平处理条件下,内参基因的表达稳定性并不相同,综合来看TUBB、β-Actin和RPS2基因的稳定性相对较好;geNorm程序在不同的GLuN水平下,依次给出了TBP/RPS2(1.08)、RPS2/β-Actin (0.88)、TBP/TUBB(1.16)和RPS2/HMBS(0.77)表达稳定性内参基因组合,由此说明TBP、β-Actin和RPS2基因的稳定性较好;NormFinder程序分析显示RPS2和HMBS基因表达稳定性较好;RefFinder在线分析软件显示,0.0％GLuN和0.8％GLuN条件下RPS2基因稳定性最高.综合本研究的分析结果,RPS2和TBP这样的内参基因适合用于产蛋后期笼养蛋鸡基因定量表达分析,而内参基因RPL4的表达稳定性最差,最不适合用于蛋鸡的定量分析内参,本实验结果对GluN处理下矫正目的基因的表达提供了理论依据.%The reference gene expression stability levels are the important factors to determine the reliability of quantitative Real-time polymerase chain reaction (qRT-PCR) results.This study was conducted to explore the selection of stable reference genes in different treatment of Glucosamine (GluN) to ensure the reliability and accuracy of gene expression analysis in caged layers (Gallus gallus domesticus) during the late laying period.A total of 500-day HY-Line layers were selected to the research objects which were randomly divided into four groups,the layers in control group were fed with the diet of the corn-soybean meal basal diet,while the others in experiment groups were fed the basal diet supplemented with 0.4％,0.6％,0.8％ GLuN,respectively.Expression of ten housekeeping genes,ribosomal protein S2 (RPS2),β-Actin,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),hydroxymethyl biliary synthetase(HMBS),TATA-box binding protein (TBP),hypoxanthine phosphoribosyltransferase 1 (HPRT1),tubulin beta class (TUBB),succinate dehydrogenase complex flavoprotein subunit A (SDHA),ribosomal protein L4 (RPL4) and beta-2 microglobulin (B2M) were assessed by qRT-PCR in 8 tissues (heart,liver,lung,kidney,duodenum,pancreas,uterus and tibia),and 3 online reference genes stability assessment tools(geNorm,NormFinder,RefFinder) and the methods of Cycle threshold (Cq) and Delta cycle threshold (Delta CT) were used to the expression stability assessment of 10 reference gene by the qRT-PCR data.The results were shown as follows.The expression stability of same candidate reference gene varied with different treatment and tissues from the analysis of Cq mean value,which explained that the 10 reference genes had a certain expression difference in these 8 tissues.Under the different GluN treatment,the method of Delta CT found that the expression stability of reference genes were not the same,in general,the expression stability of TUBB、β-Actin and RPS2 were better than other genes.From the geNorm software,TBP/RPS2(1.08),RPS2/β-Actin(0.88),TBP/TUBB(1.16) and RPS2/HMBS(0.77) showed the stable expression under different GLuN treatment,which showed that the expression stability of TBP,β-Actin and RPS2 were better.The NormFinder program software showed that RPS2 and HMBS were the stable genes.The refFinder online software showed that the RPS2 had most stable expression under 0.0％ and 0.8％ GluN group.From the comprehensive analysis of above results,RPS2 and TBP genes can apply to the quantitative expression analysis for the caged layers during the late laying period,while the expression stability of RPL4 gene was the worst,and it can not be applied to quantitative expression analysis.This experiment results also provide a theoretical basis for correcting the expression of target genes under the treatment of GluN.
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