不同GluN条件下产蛋后期笼养蛋鸡qRT-PCR内参基因的筛选

发布时间：2018-05-28 09:46

本文选题：内参基因 + qRT-PCR　；参考：《农业生物技术学报》2017年12期

【摘要】：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q RT-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ge Norm、Norm Finder、Ref Finder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;ge Norm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;Norm Finder程序分析显示RPS2和HMBS基因表达稳定性较好;Ref Finder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。
[Abstract]:The stability of internal reference gene expression will determine the reliability of real-time fluorescence quantitative PCR(Quantitative Real-time qRT-PCRR analysis. The aim of this study was to screen the stable and reliable internal reference genes of different concentrations of glucosamine gallus (GluN) in the later laying stage of cage laying hens, so as to ensure the reliability of the results of gene expression analysis in the late laying stage of laying hens. In the experiment, 500 days old Hailan grey laying hens were used as the research object, and the control group was fed with corn-soybean meal diet. In the experimental group, 0. 4% and 0. 8 GluNs were added to the basal diet, respectively. The ribosomal protein S2(ribosomal protein S2 / RPS2, 尾 -Actinine 3-glyceraldehyde-3-phosphate dehydrogenase, hydroxymethyl biliary synthetase HMBSN, telomeric binding protein TATA-box binding protein, hypoxanthine were detected by Q RT-PCR technique. Ten internal reference genes of guanine phosphate ribonucleosyltransferase (1(hypoxanthine phosphoribosyltransferase 1), tubulin beta (tubulin beta), succinate dehydrogenase complex subunit (A(succinate dehydrogenase complex flavoprotein subunit), ribosomal protein L4(ribosomal protein L4 (RPL4) and microglobulin beta-2 microglobulinB2M (B2M) were found in different tissues. The expression of liver, lung, kidney, duodenum, tibia, pancreas and uterus were analyzed, and the stability of expression of each internal reference gene was evaluated by cycle thresholdc threshold method, Delta cycle threshold method and ge NormNorm FinderRef Finder software. The results showed that the expression of the same internal reference gene was different in different treatments and tissues by using the original Cq mean value analysis. The analysis of Cq value showed that the stability of RPS2 gene expression was the best. The results of Delta CT analysis showed that under different GluN levels, the expression of RPS2 gene was more stable than that of control group. The stability of Tupb, 尾 -Actin and RPS2 genes was relatively good in different GluN levels, and the relative stability of TBP / 尾 -Actin and RPS2 genes was obtained in turn at different GluN levels, and the expression stability of TBP / 尾 -Actinine 0.88% TBP- TUBB1.16) and RPS2 / HMBS0.77) were obtained respectively, and the relative stability of Tupb, 尾 -Actin-Actin and RPS2 genes were compared with that of RPS2HMBS0.77. The results showed that the stability of RPS2, 尾 -Actin and RPS2 genes was better than that of normal Finder program. The stability of RPS2 and HMBS gene expression was better than that of Ref Finder software. The stability of RPS2 gene was the highest under the condition of GluN and 0.8%GluN. According to the results of this study, RPS2 and TBP are suitable for quantitative analysis of gene expression in cage laying hens, while RPL4 is the least stable, and is the least suitable for quantitative analysis of internal parameters in laying hens. The results of this study provide a theoretical basis for the expression of the target gene under GluN treatment.
【作者单位】：贵州大学动物科学学院;
【基金】：国家自然科学基金(No.31560642)
【分类号】：S831

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