Affiliation:
1. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
2. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China
3. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China
Abstract
The reference gene expression is not always stable under different experimental conditions, and screening of suitable reference genes is a prerequisite in quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we investigated gene selection, and the most stable reference gene for the Chinese mitten crab (Eriocheir sinensis) was screened under the stimulation of Vibrio anguillarum and copper ions, respectively. Ten candidate reference genes were selected, including arginine kinase (AK), ubiquitin-conjugating enzyme E2b (UBE), glutathione S-transferase (GST), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF-1α), α-tubulin (α-TUB), heat shock protein 90 (HSP90), β-actin (β-ACTIN), elongation factor 2 (EF-2) and phosphoglucomutase 2 (PGM2). Expression levels of these reference genes were detected under the stimulation of V. anguillarum at different times (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) and copper ions in different concentrations (11.08 mg/L, 2.77 mg/L, 0.69 mg/L and 0.17 mg/L). Four types of analytical software, namely geNorm, BestKeeper, NormFinder and Ref-Finder, were applied to evaluate the reference gene stability. The results showed that the stability of the 10 candidate reference genes was in the following order: AK > EF-1α > α-TUB > GAPDH > UBE > β-ACTIN > EF-2 > PGM2 > GST > HSP90 under V. anguillarum stimulation. It was GAPDH > β-ACTIN > α-TUB > PGM2 > EF-1α > EF-2 > AK > GST > UBE > HSP90 under copper ion stimulation. The expression of E. sinensis Peroxiredoxin4 (EsPrx4) was detected when the most stable and least stable internal reference genes were selected, respectively. The results showed that reference genes with different stability had great influence on the accurate results of the target gene expression. In the Chinese mitten crab (E. sinensis), AK and EF-1α were the most suitable reference genes under the stimulation of V. anguillarum. Under the stimulation of copper ions, GAPDH and β-ACTIN were the most suitable reference genes. This study provided important information for further research on immune genes in V. anguillarum or copper ion stimulation.
Funder
Central Public-interest Scientific Institution Basal Research Fund
Jiangsu Major New Agricultural Variety Creation
The Key Research and Development Program of Jiangsu Province
Jiangsu Revitalization of Seed Industry
Taizhou Agricultural Seed Industry Research Project
Subject
Genetics (clinical),Genetics