Cloning, identification and accurate normalization expression analysis of PPARα gene by GeNorm in Megalobrama amblycephala.

Megalobrama amblycephala suffers from serious liver diseases recently and PPARα gene has been reported to play an important role in the immune system of animal liver. On the basis of these facts, we have cloned and identified full-length cDNA of PPARα and examined its expression patterns at different embryo developmental stages and in different tissues of adult and young fish in order to improve liver disease immunity of M. amblycephala. We also accurately normalized seven reference genes by GeNorm and calculated their gene expression normalization factors. The total length of PPARα cDNA was 2021 bp, comprising of 214-bp 5'-untranslated region; 1404-bp open reading frame (encoding 467-amino acids); and 403-bp 3'-untranslated region. PPARα peptide was predicted to consist of 4 domains, i.e. A/B, C, D, and E/F. PPARα mRNAs were detected in different tissues of adult and young fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, the expression of PPARα in white muscles was highest followed by liver and it was lowest in gonads. Its expression in male gonads was significantly higher than female gonads. In young fish, the expression of PPARα was highest in brain, followed by intestines and it was lowest in spleen. At different embryo developmental stages, the expression of PPARα was highest at 2 cells stage and it was lowest at gastrula stage, but it increased on first day after hatching. In unfertilized spermatozoa, the expression of PPARα was higher than unfertilized ovum.