Cloning, Expression and Stability Analysis of Candidate Reference Genes in the Glacial Relict <i>Hucho bleekeri</i> Under Immune Stress

To find reference genes (RGs) and establish a screening method for RGs in Sichuan taimen ( Hucho bleekeri ), six candidate RGs— β-actin , b2m , β-tubulin , ef1α , gapdh and hprt — were cloned, and the stability of expression of the six candidate RGs were analysed. The six RG sequences in H. Bleekeri had higher similarity with the corresponding sequences in Salmo or Oncorhynchus . The RGs geomean of ranking values of gene expression in tissues of H. bleekeri was ef1α &gt; β-actin &gt; β-tubulin &gt; hprt &gt; b2m &gt; gapdh . After lipopolysaccharide (LPS) treatment, the RGs geomean of ranking values of gene expression in spleen was ef1α &gt; β-actin &gt; b2m &gt; gapdh &gt; β-tubulin &gt; hprt , in head kidney was β-actin &gt; ef1α &gt; β-tubulin &gt; hprt &gt; gapdh &gt; b2m , in liver was β-tubulin &gt; hprt &gt; β-actin &gt; gapdh &gt; ef1α &gt; b2m ; after polyinosinic: polycytidylic acid (Poly (I:C)) treatment, the values in spleen was ef1α &gt; hprt &gt; β-actin &gt; b2m &gt; β-tubulin &gt; gapdh , in head kidney was β-actin &gt; ef1α &gt; β-tubulin &gt; hprt &gt; gapdh &gt; b2m , in liver was β-tubulin &gt; β-actin &gt; ef1α &gt; gapdh &gt; b2m &gt; hprt . The results indicated that ef1α can be used as the most stable RG for the tissue distribution analysis of gene expression in H. bleekeri , and ef1α, β-actin and β-tubulin was suitable for spleen, head kidney and liver cells, respectively, under LPS and Ploy (I: C) stimulation.