Screening of Suitable Reference Genes for Immune Gene Expression Analysis Stimulated by Vibrio anguillarum and Copper Ions in Chinese Mitten Crab (Eriocheir sinensis)
文献类型： 外文期刊
作者： Yan, Fengyuan 1 ; Li, Hui 1 ; Chen, Xue 1 ; Yu, Junjie 2 ; Su, Shengyan 1 ; Li, Jianlin 2 ; Ye, Wei 2 ; Tang, Yongkai 1 ;
作者机构： 1.Shanghai Ocean Univ, Natl Demonstrat Ctr Expt Fisheries Sci Educ, Shanghai 201306, Peoples R China
2.Nanjing Agr Univ, Wuxi Fisheries Coll, Wuxi 214081, Peoples R China
3.Chinese Acad Fishery Sci, Freshwater Fisheries Res Ctr, Key Lab Freshwater Fisheries & Germplasm Resources, Minist Agr & Rural Affairs, Wuxi 214081, Peoples R China
关键词： Chinese mitten crab; reference gene; qRT-PCR; Vibrio anguillarum; copper ion
期刊名称：GENES （ 影响因子：3.5； 五年影响因子：3.9 ）
ISSN：
年卷期： 2023 年 14 卷 5 期
页码：
收录情况： SCI
摘要： 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-3phosphate dehydrogenase (GAPDH), elongation factor 1 alpha(EF-1 alpha), alpha-tubulin (alpha-TUB), heat shock protein 90 (HSP90), beta-actin (beta-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-1 alpha > alpha-TUB > GAPDH > UBE > beta -ACTIN > EF-2 > PGM2 > GST > HSP90 under V. anguillarum stimulation. It was GAPDH > beta-ACTIN > alpha-TUB > PGM2 > EF-1 alpha > 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-1 ff were the most suitable reference genes under the stimulation of V. anguillarum. Under the stimulation of copper ions, GAPDH and b-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.
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