摘要
内参基因在实时荧光定量PCR(qRT-PCR)中具有校准的作用,然而鲤疱疹病毒Ⅱ型感染异育银鲫内参基因目前仍未见研究报道。采用qRT-PCR技术检测不同处理条件下异育银鲫组织和尾鳍细胞GAPDH、EF-1α、18S rRNA和β-actin 4个候选内参基因在组织和尾鳍细胞的转录水平,利用软件geNorm、Norm Finder、Best Keeper和Delta Ct分析了其表达量的稳定性,以筛选出健康异育银鲫不同组织以及鲤疱疹病毒Ⅱ型感染的肾脏、脾脏和异育银鲫尾鳍细胞在不同时间均较稳定的内参基因。geNorm稳定值以及4个内参的表达量Ct值分析显示,健康异育银鲫脑、脾脏、肾脏、肌肉、鳃、肠、肝脏和心脏组织中β-actin和EF-1α都是比较稳定的内参基因;鲤疱疹病毒Ⅱ型感染肾脏和尾鳍细胞不同时间点,内参基因β-actin稳定性最佳;鲤疱疹病毒Ⅱ型感染脾脏不同时间点时,EF-1α稳定性最佳。分别以4个候选内参基因为内参分析PIN1基因在肾脏组织不同感染时间的相对表达量,结果进一步证实,PIN1基因在肾脏组织感染不同时间点以β-actin为内参时,其表达量呈下降趋势,与cDNA文库测序中的表达量分析结果一致,各时间点的表达量差异极显著(P<0.01)。本试验结果有利于研究异育银鲫在不同处理条件下基因的表达分析,可为获得精准的结果奠定理论基础。
Reference genes can be calibrated by real-time fluorescence quantitative PCR(qRT-PCR).However,there is still no study on the infection of allogynogenetic silver crucian carp Carassius auratus gibelio with Cyprinid herpesvirusⅡ(CyHV-2).In this study,the transcription levels of GAPDH,EF-1α,18 SrRNA andβ-actin in tissues and caudal fin cells(GiCF)of allogynogenetic silver crucian carp under different treatment conditions were detected by qRT-PCR,and the stability of their expression levels was analyzed by software geNorm,Norm Finder,Best Keeper and Delta Ct to screen stable Reference genes in different tissues of healthy allogynogenetic silver crucian carp,and in kidneys,spleens and caudal fin cells of allogynogenetic silver crucian carp infected with CyHV-2 at different times.The geNorm stability value and Ct value of the expression of four reference genes based on the four software analyses showedβ-actin and EF-1αwere suitable reference genes in the brain,spleen,kidney,muscle,sputum,intestine,liver and heart tissues;that theβ-actin gene was stable reference gene in kidney and GiCF cells at different time points underlying CyHV-2 infection;and that the EF-1αwas stable reference gene in spleen at different time underlying CyHV-2 infection.The relative expression levels of PIN1 gene were analyzed in kidney at different time using four candidate reference genes as internal parameters to further confirm the above results.It was found that the expression trend of the PIN1 gene was decreased in at different time in the kidney infection usingβ-actin as the reference,which was more consistent with the sequencing and expression analysis of the cDNA library,and the expression was significantly different at each time(P<0.01),indicating that the findings are conducive to the study of gene expression analysis and to provide theoretical foundation for obtaining accurate results underlying different treatment conditions.
作者
费越越
南星羽
余路
罗扬
高钟元
许丹
FEI Yueyue;NAN Xingyu;YU Lu;LUO Yang;GAO Zhongyuan;XU Dan(National Pathogen Collection Center for Aquatic Animals, Shanghai 201306, China;Key Laboratory of Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China;National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China)
出处
《水产科学》
CAS
CSCD
北大核心
2020年第3期306-315,共10页
Fisheries Science
基金
国家自然科学基金青年基金资助项目(31602203).
