PPARγ, an important gene related to lipid metabolism and immunity in Megalobrama amblycephala: cloning, characterization and transcription analysis by GeNorm.

In order to be able to modulate and improve the function of PPARγ and decrease further some metabolic diseases of M. amblycephala, we have cloned and identified the full-length cDNA of PPARγ in M. amblycephala and examined its transcription patterns at different embryo developmental stages and in different tissues of adult and immature fish. We also accurately normalized seven reference genes by GeNorm and calculated their gene transcription normalization factors. The full-length of PPARγ was 1968 bp, consisting of 218 bp 5'-untranslated region, 1,533 bp open reading frame encoding 510 amino acids residues and 217 bp 3'-untranslated region. M. amblycephala PPARγ peptide was predicted to consist of 4 conserved domains, i.e. N-terminal domain, DNA-binding domain, ligand binding domain and flexible hinge region. PPARγ mRNAs were detected in all studied tissues of adult and immature fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, PPARγ transcription in liver was highest, followed by gills and it was lowest in female gonads. Moreover, the differences among liver, gill, intestine/brain, spleen/white muscle, kidney and female gonads were greatly significant (p<0.01). The transcription of PPARγ in male gonads was significantly higher than in female gonads (p<0.01). In immature fish, the transcription of PPARγ was highest in intestines followed by adipose tissue, and it was lowest in hearts and white muscles. A great difference was observed (p<0.01) in the transcription of PPARγ among adipose tissue, intestines, liver and heart/white muscles. At different embryo developmental stages, PPARγ transcription in unfertilized spermatozoa was greatly higher than in unfertilized ovum (p<0.01) and it was highest among different embryo developmental stages. The transcription of PPARγ increased gradually during 2 cells stage and 32 cells stage and then decreased until gastrula stage at which it was lowest. The transcription of PPARγ increased again on first day after hatching. There was a significant difference (p<0.01) in the transcription of PPARγ between 2 cells stage and 32 cells stage and it was same between 32 cells stage and gastrula stage. These results revealed that transcription of PPARγ showed a tissue-dependent regulation and a developmental-stage-dependent regulation that are valuable and helpful to improve the function of PPARγ and to decrease some metabolic diseases in the culture of M. amblycephala.