实时荧光定量PCR检测皱纹盘鲍Δ5脂肪酸去饱和酶基因表达方法的建立及应用

摘要

研究以皱纹盘鲍(Haliotis discus hannai Ino)体内存在的2条Δ5 Fad (Hdhfad1和Hdhfad2)为目的基因,β-肌动蛋白(β-actin)和核糖体蛋白S9(Ribosomal protein S9, RPS9)为内参基因,应用2-ΔΔCt方法建立了实时荧光定量PCR (Real-Time quantitative PCR, RT-qPCR)检测体系,并应用此体系分析了鲍鱼肌肉组织Δ5 Fad在不同饲料处理下的表达差异。实验饲料含有不同的脂肪源,分别是棕榈酸甘油酯(Tripalmitin, TP 饲料)、富含二十碳四烯酸(Arachidonic acid, ARA)的油脂(AO饲料)和富含二十碳五烯酸(Eicosapentaenoic acid, EPA)的油脂(EO 饲料)。结果表明：针对 Hdhfad1、Hdhfad2、β-actin 和 RPS9所设计的引物特异性强。各引物对的PCR 扩增效率(Efficiency, E)分别为1.05、0.99、0.97和0.98,满足2-ΔΔCt方法对 E 的要求。当退火温度为52℃,反应体积为25µL时, RT-qPCR的扩增效果最好。所建立的体系能够准确定量Δ5 Fad的基因表达。利用该方法分析Δ5 Fad在不同饲料处理下的表达结果显示,与TP对照组相比, EO和AO饲料显著降低了鲍鱼肌肉组织Δ5 Fad(Hdhfad1和Hdhfad2)的表达量。%Δ5 fatty acyl desaturase (Fad) is the key enzyme for the biosynthesis of long chain polyunsaturated fatty acids. Abalone, Haliotis discus hannai Ino, has two genes of Δ5 Fad (Hdhfad1 and Hdhfad2), of which the cDNA se-quences are highly similar (96.82%). In this study, an efficient Real-Time quantitative RCR (RT-qPCR) assay based on the 2-ΔΔCt method was established for measuring the expression levels of Hdhfad1 and Hdhfad2. Data were normalized to the gene expression levels ofβ-actin and ribosomal protein S9 (RPS9). The effects of different dietary lipids on the ex-pressions of Hdhfad1 and Hdhfad2 were studied in muscle tissue of abalone using this assay. We prepared three types of diets that contain different dietary lipids-tripalmitin (TP diet), ARA oil (AO diet) and EPA oil (EO diet). The results showed that primers designed for Hdhfad1, Hdhfad2,β-actin and RPS9 were specific, with the amplifying efficiency of 1.05, 0.99, 0.97 and 0.98 respectively. The efficiency of these primers was suitable for the 2-ΔΔCt method. The optimal annealing temperature and reaction volume for the RT-qPCR amplification were 52℃ and 25 µL respectively. We con-cluded that this assay was rapid and sensitive in analyzing the expressions of Δ5 Fad in abalone. This assay demon-strated that the expression levels of Hdhfad1 and Hdhfad2 were significantly decreased in abalone fed with EO or AO diet compared to those of TP group.