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| Screening of Reference Genes in Allogynogenetic Silver Crucian Carp Carassius auratus gibelio |
| FEI Yueyue1,2, NAN Xingyu1,2, YU Lu1,2, LUO Yang1,2, GAO Zhongyuan1,2, XU Dan1,2,3 |
1.National Pathogen Collection Center for Aquatic Animals, Shanghai 201306, China;
2.Key Laboratory of Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China;
3.National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China |
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Abstract 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 herpesvirus Ⅱ (CyHV-2). In this study, the transcription levels of GAPDH, EF-1α, 18 SrRNA and β-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 showed β-actin and EF-1α were suitable reference genes in the brain, spleen, kidney, muscle, sputum, intestine, liver and heart tissues; that the β-actin gene was stable reference gene in kidney and GiCF cells at different time points underlying CyHV-2 infection; and that the EF-1α was 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 using β-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.
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Received: 12 April 2019
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