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| Cloning and Expression Stability of Reference Genes β-actin, GAPDH and EF1-α in Acipenser dabryanus |
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Abstract Dabry's sturgeon (Acipenser dabryanus) is a national-level protected animal with important ecological value and of germplasm resources value. Molecular biology techniques such as real-time quantitative real-time PCR (qRT-PCR) play an important role in the protection and germplasm development process of Dabry's sturgeon. The selection of internal reference genes in qRT-PCR technology is related to the accuracy of the test results. In order to screen the potential reference gene of Dabry's sturgeon, Three potential internal reference genes, namely β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1α (EF1-α), were obtained by homologous cloning combined with local Blast method. Mega 5.05, DNAstar 7.1, CPHmodels 3.2, and ClustalW were used to analyze the biological information of this three genes. The qRT-PCR was used to detect the three gene Cq value in the brain, liver, kidney, pancreas, spleen, esophagus, stomach, pyloric caeca, duodenum,intestinum valula, rectum, muscle, gill and skin of Dabry's sturgeon. The expression stability of the three genes in 14 tissues was evaluated using geNorm, NormFinder and Bestkeeper. The results showed that the full-length CDS of potential qRT-PCR reference genes β-actin, GAPDH and EF1-α were 1125, 999 and 1383 bp, encoding 375, 333 and 461 amino acids, respectively. Bioinformatics analysis showed that the amino acid sequence and protein
three-dimensional structure deduced by the three genes were highly consistent with other species. The amplification efficiencies of β-actin, GAPDH and EF1-α qRT-PCR primer, was 92.2%, 93.0%, and 92.7%, respectively; the melting temperature (Tm) of the amplification products were 84°C, 83.5°C, and 85°C, respectively. Comprehensive
geNorm, NormFinder and Bestkeeper evaluation results showed that the stability of the three reference genes was
EF1-α>β-actin>GAPDH in 14 tissues and EF1-α and β-actin can be used as reference genes for Dabry's sturgeon qRT-PCR study. In this experiment, we obtained the full-length CDS of three potential reference genes β-actin, GAPDH and EF1-α, and confirmed that EF1-α and β-actin are suitable for the qRT-PCR study of Dabry's sturgeon. This study provides basic data for the application of qRT-PCR technology in Dabry's sturgeon research.
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Received: 10 May 2018
Published: 24 October 2018
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