Transcriptional Regulation Mechanisms And Functional Molecular Marker Identification Of PCK1Gene

Phosphoenolpyruvate carboxykinase1（PCKl）, also named PEPCK-C, is a multiple-functions gene which is involved in gluconeogenesis, glyceroneogenesis, reproduction and female fertility, the development of obesity and diabetes. And the last research indicated that the facility and reproduction span of transgenic PCK1mouse were longer than normal mouse. This research is a preliminary study on the PCK1gene expression and Molecular mechanism from three aspects of identification and expression analysis of alternative splicing isoforms, the core promoter region determination, microRNA targets and its function identification and is described in the following three sections. Section1Alternative splicing and mRNA expression analysis of bovine PCK1geneThe mRNA expression and possible splice variants of the PCK1were investigated by screening its cDNA in9tissues （heart, live,spleen, lung, kidney, skeletal muscle, ovary, testis and sperm） in Holstein bull and cow. PCK1mRNA was highly expressed in the live, kidney, ovary and testis, low in the heart, spleen and lung tissues, while it was not detected in the skeletal muscle, sperm. This led to the discovery of5novel bovine splice variants, named PCK1-AS1～PCK1-AS5. These splice variants potentially encoded for a shorter protein （605AA,546AA,373AA,246AA and246AA, respectively） as compared to the complete protein （622AA）. The PCK1-AS1～PCK1-AS5splicing isomers exist different degrees of functional domains missing or lost, resulting in weakening or loss of the PCK1gene function encoding. Section2Cloning and identification of core promoter area of bovine PCK1geneIn order to investigate the molecular mechanisms of the gene expression regulation in bovine PCK1gene, the promoter region of bovine PCK1gene was predicted by relative online software. A series of expression vectors which contains the deletion fragments of PCK1promoter fragments were constructed, the sequences were analyzed in HepG2cell lines. In this study, luciferase reporter gene system and PCR-RFLP methods were used to research the activity of promoter in a series of fragments of the bovine PCK1gene untranslated region to find its core promoter region. Finally, the core promoter region of bovine PCK1gene was determined at g.-785～g.-462. This test laid the foundation to study PCK1gene in the molecular mechanisms of post-transcriptional regulation region. Section3Expression analysis of the bovine the PCK1gene targets microRNAsAnalysis of the3’UTR of the PCK1mRNA by RT-PCR and clone sequence technology showed that there has two novel SNPs （SNP1and SNP2corresponding to nt2108and2183of Genbank Accession number NM₁74737, respectively）, and the SNP2position of the PCK1mRNA3’UTR contains a sequence that matches the bta-miR-26a seed region which can significantly reduce the expression of PCKl. This is the first study to investigate the role both the promoter and3’UTR in regulating PCK1expression and our results provide a systematic view of gene regulation in the bovine, which will be refined as additional mammalian genomes become available. The target binding of bta-miR-26a and the PCK1gene3’flanking region, the binding situation is controlled by the type of SNP, is affecting the expression of PCKlgene which may cause the cows reproductive performance aspects difference. bta-miR-26a significantly inhibited PCK1gene expression after the mutation, the mutation sites can be used as a potential functional molecular markers of cow reproductive traits.