青海湖裸鲤实时荧光定量PCR内参基因筛选与验证

Selection and Validation of Reference Genes in Gymnocypris przewalskii by Quantitative Real-Time PCR

为探明青海湖裸鲤候选内参基因在低温环境下的表达稳定性,根据RNA-seq定量结果筛选表达稳定的候选内参基因,利用实时荧光定量PCR检测候选基因在0℃和17℃下,脑、肝胰脏、肌肉组织中的表达量,并通过GeNorm、Norm Finder和Best Keeper3种分析程序分析其表达稳定性,筛选出理想的内参基因。结果显示:根据RNA-seq定量结果,共筛选获得脱水酶(Abhd18)基因、微球蛋白(B2m)基因、延伸蛋白A(Eloa)基因、DAZ相关蛋白2(Dazap2)基因、肌动蛋白(Actb)基因、磷酸甘油醛脱氢酶(Gapdh)基因、BCL细胞死亡激动因子(Bad)基因和脂肪酸结合蛋白3(Fabp3)基因等8个候选基因;对上述基因的表达稳定性进行分析,发现表达最稳定的是B2m和Eloa基因,表达最不稳定的是Fabp3基因;利用B2m和Eloa基因作为双内参基因对4个脂肪酸代谢相关基因在0℃和17℃脑、肝胰脏、肌肉组织中的表达量进行校正,其相对表达比值与RNA-seq结果高度正相关。研究结果可为高原鱼类极端环境适应的研究提供候选内参基因。

In order to clarify the expression stability of the candidate reference gene of Gymnocypris przewalskii under low temperature environment,120-day-old G.przewalskii with body weight of(8.5±1.2)g were randomly divided into low temperature group(reared at 17℃for one week,and then cooled to 0℃at a rate of 0.5℃/h,and kept at 0℃for 15 d)and the control group(17℃constant temperature).Then,according to the quantitative results of RNA-seq,the candidate internal reference genes with stable expression were screened,and real-time quantitative PCR(qRT-PCR)was used to detect the expression levels of the candidate genes in brain,hepatopancreas,and muscle tissue at 0℃and 17℃.Then the expression stabilities of reference genes were analyzed by GeNorm,Norm Finder and Best Keeper procedures.The RNA-seq analysis revealed that 8 genes were obtained as candidate reference genes including dehydrase(Abhd18)gene,microglobulin(B2m)gene,extension protein A(Eloa)gene,DAZ related protein 2(Dazap2)gene,actinb(Actb)gene,phosphoglyceraldehyde dehydrogenase(Gapdh)gene,BCL cell death agonist(Bad)gene and fatty acid binding protein 3(Fabp3)gene.The expression stability analysis of the 8 candidate genes showed that B2m and Eloa were the most stable candidate genes,and Fabp3 was the most unstable candidate gene.B2m and Eloa were used as double reference genes to correct the expression levels of four fatty acid metabolity-related genes in brain,hepatopancreas and muscle at 0℃and 17℃,and the results showed that the relative expression ratios of qRT-PCR were highly positively correlated with RNA-seq results.The finding provides candidate reference genes for the researches on extreme environmental adaptation of plateau fishes.