产蛋前期和产蛋期籽鹅组织内参基因的稳定性

摘要

[目的]分析比较使用广泛的7个候选内参基因在产蛋前期和产蛋期籽鹅组织中的表达情况,筛选籽鹅组织基因表达分析中最佳的内参基因及其数目.[方法]应用实时定量反转录PCR技术,分别检测28S rRNA、18S rRNA、GAPDH、ACTB、HPRT1、SDH和TUB在产蛋前期与产蛋期健康籽鹅肝脏、肾脏、心脏、腿肌和卵巢组织中的表达情况,采用绝对定量方法确定基因拷贝数,然后分别使用geNorm和NormFinder程序进行数据分析.[结果]产蛋前期籽鹅各组织中7个内参基因表达稳定度的顺序分别为:GAPDH=HRPT1(0.195) ＞TUB(0.244)＞28S rRNA(0.414) ＞18S rRNA(0.495) ＞ACTB (0.541) ＞SDH (0.610);产蛋期籽鹅各组织中7个内参基因表达稳定度的顺序分别为:GAPDH=28S rRNA(0.128)＞TUB(0.181) ＞ACTB(0.192) ＞SDH(0.221) ＞HRPT1(0.316)＞18S rRNA(0.362),且7个基因的配对差异分析分别为V2/3(产蛋前期)-0.084、V2/3(产蛋期)-0.069,内参基因的最适数目均为2个.[结论]产蛋前期和产蛋期籽鹅组织目的基因的表达分析中相对适合的内参基因分别为GAPDH和HRPT1、GAPDH和28S rRNA.%[Objective] The mRNA expression levels of seven candidate reference genes in green-goose tissues during prelaying and laying periods were comparatively-analyzed. Finally the most appropriate reference genes and their numbers were screened in gene expression analysis of green-goose tissues. [ Method] The expressions of candidate reference genes 28S rRNA, 18S rRNA, GAPDH, ACTB, HPRT1, SDH and TUB in prelaying and laying green-goose liver, kidney, heart, leg muscle and ovary were detected by qRT-PCR. Gene copy number was determined using absolute quantification. Then the expression stability of these genes was analysed by geNorm software. [Result] The results showed that stability of the seven reference genes from high to low was GAPDH=HRPT1 (0.195)>TUB (0.244)>28S rRNA(0.414)>18S rRNA (0.495)>ACTB (0.541)>SDH (0.610) during prelaying period and GAPDH=28S rRNA (0.128)>TUB (0.181)>ACTB (0.192)>SDH (0.221)>HRPTl (0.316)>18S rRNA (0.362) during laying period by geNorm analysis. And V2/3 of pairwise variance analysis of 7 candidate reference genes were separately 0.084 and 0.069 during the 2 periods. The appropriate number of reference genes was 2. [ Conclusion ] These results suggest that GAPDH and HRPT1, GAPDH and 28S rRNA are suitable reference genes and could be used to normalize mRNA levels between different samples in green-goose tissues during prelaying and laying periods.