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| Selection and Validation of Reference Genes in Gymnocypris przewalskii by Quantitative Real-Time PCR |
| LIU Sijia1, QI Delin2, QI Hongfang3, HAN Buying1, NIU Yimeng1, ZHAO Kai1, TIAN Fei1 |
1. Qinghai Key Laboratory of Animal Ecological Genomics, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China;
2. State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China;
3. Key Laboratory of Breeding and Protection of Gymnocypris przewalskii in Qinghai Province, Rescue Center of Gymnocypris przewalskii, Xining 810016, China |
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Abstract In order to clarify the expression stability of the candidate reference gene of Gymnocypris przewalskii under low temperature environment, 120-day-old G. przewalskii with body weight of (8.5±1.2) g were randomly divided into low temperature group (reared at 17 ℃ for one week, and then cooled to 0 ℃ at a rate of 0.5 ℃/h, and kept at 0 ℃ for 15 d) and the control group (17 ℃ constant temperature). Then, according to the quantitative results of RNA-seq, the candidate internal reference genes with stable expression were screened, and real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of the candidate genes in brain, hepatopancreas, and muscle tissue at 0 ℃ and 17 ℃. Then the expression stabilities of reference genes were analyzed by GeNorm, Norm Finder and Best Keeper procedures. The RNA-seq analysis revealed that 8 genes were obtained as candidate reference genes including dehydrase (Abhd18) gene, microglobulin(B2m) gene, extension protein A (Eloa) gene, DAZ related protein 2(Dazap2) gene, actinb(Actb) gene, phosphoglyceraldehyde dehydrogenase (Gapdh) gene, BCL cell death agonist (Bad) gene and fatty acid binding protein 3 (Fabp3) gene.The expression stability analysis of the 8 candidate genes showed that B2m and Eloa were the most stable candidate genes, and Fabp3 was the most unstable candidate gene.B2m and Eloa were used as double reference genes to correct the expression levels of four fatty acid metabolity-related genes in brain, hepatopancreas and muscle at 0 ℃ and 17 ℃, and the results showed that the relative expression ratios of qRT-PCR were highly positively correlated with RNA-seq results. The finding provides candidate reference genes for the researches on extreme environmental adaptation of plateau fishes.
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Received: 28 May 2021
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