DNA Cloning、Expression And DNA Methylation Status Analysis Of Landes Geese C/EBP Gene

Primers were designed for geese according to the C/EBPβ gene sequence of chicken, Zebra Finch, Quail, human, and hamster in the Genebank. the total coding region of lande goose C/EBPa and C/EBPP gene were obtained and submitted to GenBank (accession number:GU289824and GU068582). Bioinformatics methods are adopted to predict the structure and function of C/EBPa and C/EBPP protein, and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species. To check relative expression levels of C/EBPP mRNA in various porcine tissues, the real-time PCR was performed. And determined the effects of dietary betaine supplementation on the mRNA expression level of C/EBPβ in landes goose fatty liver.1. DNA cloning and sequencing of goose C/EBPa and C/EBPβ:C/EBPa gene of Landes geese was cloned. Its DNA was1401bp in length, had been submitted to GenBank as GU289824. C/EBPβ gene of Landes geese was cloned. Its DNA was2130bp in length, had been submitted to GenBank as GU068582.2. Bioinformatics analysis of C/EBPa and C/EBPβ protein:Using bioinformatics network resources and relevant softwares, we predict that C/EBPa DNA has a975bp open reading frame (ORF) flanked by a210bp5’UTR and a397bp3’UTR. The deduced amino acid sequence has324residues, which contains no signal peptide, typical hydrophobic regions and transmembrane region, but is a nucleoprotein residing in nucleolus. A Conserved Domain Database alignment reveals that C/EBPa protein contains a carboxyl-terminal basic-leucine zipper (bZIP) domain, which are characterized by cysteine-rich, double-zinc finger motifs and a diatinct homeodomain.We predict that C/EBPβ DNA has a984bp open reading frame (ORF). The deduced amino acid sequence has324residues, which contains no signal peptide, typical hydrophobic regions and transmembrane region, but is a nucleoprotein residing in nucleolus. A Conserved Domain Database alignment reveals that C/EBPP protein contains a carboxyl-terminal basic-leucine zipper (bZIP) domain, which are characterized by cysteine-rich, double-zinc finger motifs and a diatinct homeodomain.3. RT-PCR analysis of mRNA tissue-specific expression profiles: To check the relative expression levels of C/EBPβ mRNA in various goose tissues, real-time PCR was performed. The results showed that C/EBPP gene was expressed broadly in various tissues, but at very different levels. The expression levels of this gene are higher in the liver, adipose tissue, and lung than that in other tissues, showed that the express of gC/EBPβ mRNA in lipid metabolism important organs (adipose tissue and live) has a high level. Indicates that it related with lipid metabolism closely.4. RT-PCR analysis of the effects of dietary betaine supplementation on the mRNA expression level of C/EBPβ in landes goose fatty liver.:The expression of this gene in the overfed geese was higher than that in the geese that were fed the control diet. Compared to the overfeeding groups, the geese that were fed the betaine diet showed decreased the expression of C/EBPβ mRNA in the fatty livers.5. BSB analysis of the effects of dietary betaine supplementation on the DNA methylation level of C/EBPβ in landes goose fatty liver.:We performed sodium bisulfite sequencing of individual alleles of this region, which contains54CpG dinucleotides of C/EBPβ. In the overfed group, the average methylation at the54CpGs sites were2.96%in contrast to1.48%in control group. Compared to the overfeeding group, betaine treatment (2.23%) of the average methylation at these CpGs sites can not achieve the statistic significant change. But the level of methylation in this region was decreased.