夏眠期仿刺参肠组织实时荧光定量PCR分析中内参基因的筛选

Selection of Reference Genes for Quantitative Real-Time PCR of Apostichopus aponicus Intestine During Different Periods of Aestivation

在仿刺参(Apostichopus japonicus)正常时期、夏眠初期和中期,应用实时荧光定量PCR技术,检测CA043、dpolm、ro60、grb2、hiat1、nlrc4、farp1、cyc和gapdh9个候选内参基因在肠组织中的表达稳定性。利用DeltaCt、GeNorm、NormFinder和BestKeeper 4种程序进行统计分析。发现上述条件中,9个候选内参基因在上述条件中的表达稳定性存在一定差异,不同的软件处理分析,得到的基因稳定性排序不完全一致。综合4种程序的方法,筛选出基因grb2表达最稳定,可用作内参基因,其次为基因ro60及dpolm,而表达最不稳定的是CA043,不适宜作为内参基因。本研究为仿刺参中基因表达定量分析奠定了基础,同时为仿刺参中内参基因的选择提供一定参考依据。

Apostichopus japonicus is the most important economic species in China.Its special physiological habit is aestivation.There have been many studies on breeding,development,immunology and others,molecular biological research has become important.Domestic reference gene-related research is more in higher plants and animals than in aquatic animal.The study on the stability of reference gene in aestivation has not been reported.From transcriptome data analysis,we selected nine candidate reference genes.In normal times,and at beginning of aestivation and in the mid of aestivation,With quantitative real-time PCR,we assessed the expression stability of nine candidate reference genes（CA043,dpolm,ro60,grb2,hiat1,nlrc4,farp1,cyc and gapdh）in intestinal tissues of A.japonicus.The stability of nine candidate reference genes analyzed by the 4procedures（Delta Ct,GeNorm,NormFinder and BestKeeper）.In conclusion,through the different software,the stability of candidate reference genes were not entirely consistent.The most stable gene was grb2,which can be used as the reference.The stability of ro60 and dpolm was the second;and the least stable was CA043.This study was the first to evaluate and analyze the expression stability of candidate reference genes in aestivation,laid a foundation for the further quantitative analysis of gene expression in A.japonicus.