花鲈病毒感染后qPCR内参基因的筛选与应用

Screening and application of internal reference genes for qPCR post Lateolabrax maculatus iridovirus infection in Lateolabrax maculatus

在开展花鲈(Lateolabrax maculatus)抗病毒饲料评价而采用qPCR进行病毒增殖以及免疫基因表达分析中需要首先确定病毒感染后稳定的内参基因。本研究选取12个组织/细胞(中肠、肌肉、胃、脑、心、肝、鳃、头肾、后肾、胸鳍、脾和花鲈肾细胞),采用qPCR方法检测花鲈虹彩病毒(Lateolabrax maculatus iridovirus,LMIV)感染前后RNAPoLⅡ、HPRT、TUBA、ACTB、18S rRNA、B2M、RPL7及EF1A 8个基因的Ct值,采用GeNorm、NormFinder、BestKeeper软件评估内参基因表达的稳定性。以筛选的内参基因为基础建立qPCR方法,在LMIV感染花鲈肾细胞系10、24、48、72、96、120 h后对病毒ORF92R、ORF101R、ORF407R、ORF543R基因进行表达分析。GeNorm软件结果表明8个候选内参基因在感染LMIV后的花鲈不同组织/细胞中的表达稳定性为:RNAPoLⅡ=HPRT>RPL7>TUBA>ACTB>18S rRNA>B2M>EF1A。NormFinder软件结果显示18S rRNA在对照组花鲈不同组织/细胞中表达稳定性最高;LMIV感染后RPL7是表达最稳定的内参基因。BestKeeper软件结果表明RPL7、RNAPoLⅡ、HPRT为花鲈感染LMIV后最佳参考基因组合。病毒基因的表达分析结果显示,RPL7、RNAPoLⅡ作为筛选出的稳定内参基因在分析病毒基因表达中优于18S rRNA。RPL7、RNAPoLⅡ、HPRT可作为评价花鲈感染LMIV后免疫相关基因表达以及病毒增殖的最佳qPCR参考基因,应用于花鲈抗病毒饲料的分析。

To evaluate the antiviral feed of the sea bass(Lateolabrax maculatus),this study screened the stable internal reference genes by qPCR and determined the virus proliferation and immune gene expression analysis with virus infection.In this study,8 candidate reference genes(RNAPoLⅡ,HPRT,TUBA,ACTB,18S rRNA,B2M,RPL7 and EF1A)were analyzed in 12 tissues/cells(midgut,muscle,stomach,brain,heart,liver,gills,head kidney,metanephros,pectoral fin,spleen and sea bass kidney line)of sea bass by qPCR,and used GeNorm,NormFinder,BestKeeper to evaluate the stability of internal reference gene expression.Established qPCR methods with the selected internal reference genes was to analyze the expression of the virus ORF92R,ORF101R,ORF407R,ORF543R genes with cell lins of LMIV infection at 10,24,48,72,96,120 h.The results of GeNorm software showed that the expression stability of 8 candidate internal reference genes in different tissues/cells of seabass with LMIV infection was:RNAPoLⅡ=HPRT>RPL7>TUBA>ACTB>18S rRNA>B2M>EF1A.The results of NormFinder showed that the expression stability of 18S rRNA was the highest in different tissues/cells of the control group andRPL7 was the most stable internal ref⁃erence gene.The results of BestKeeper showed thatRPL7 ,RNAPoL Ⅱ , andHPRT are the best reference gene index in infected sea bass with LMIV.RPL7 andRNAPoL Ⅱ as the stable internal reference genes were superior to 18S rRNA in terms ofanalyzing viral genes expression.RPL7 ,RNAPoL Ⅱ ,HPRT as the best qPCR reference genes can beused to evaluate immune-related genes expression and virus proliferation in sea bass, and can be ap⁃plied to analyze the antiviral of sea bass feed.