Molecular Cloning Of Geese Insulin Signaling Pathway Related Three Genes And Effects Of Glucose And Insulin On Their Expression

The pleckstrin homology domain-interacting protein (PHIP) plays important roles in insulin-mediated signal transduction pathways, promotiong GLUT4 translocation and mediating cell apoptosis and proliferation. However, the IRS1 and TBC1D1 play essential roles in insulin signal pathway, and therefore these two genes are important to the insulin mediating signal pathway transduction. In the present study, IRS1 and TBC1D1 partial mRNA sequence of goose were cloned. And the full-lenth of PHIP mRNA sequence of goose was cloned using RACE technical.Then the different concentration of glucose and insulin were added to the medium of geese primary hepatocytes cultured in vitro, to investigate their roles on mRNA expression of PHIP, IRS1 and TBC1D1 using Real-Time RT-PCR. The results were as follows:(1) The partial coding domain sequence (CDS) of IRS1, TBC1D1 genes were amplified by RT-PCR, and their length were 530bp and 300bp, respectively. The phylogenetic tree of IRS1 and TBCIDI gene in vertebrates were built using the MEGA 5.0 software, and results showed that the geese IRS1 and TBC1D1 gene have higher homology with that of Gallus, Turkey and Taenioygia guttata.(2) Three alternatively spliced forms of PHIP mRNA were found by RACE technical, and named them as PHIP-a, PHIP-b and PHIP-c, respectively. The full-lenth of total three PHIP mRNA sequeces was 5,525bp,5,080bp and 4,677bp, respectively. In silicon analysis showed that the splicing mode of all three PHIP mRNA sequences was the selective termination.(3) All the three splicing PHIP mRNA have the same CDS,encoding the same protein with 1295 amino acid. The relative molecular mass predicted is 147002.48Da, and the isoelectricpoint of PHIP protein was 8.01. PHIP amino acid sequence pocesses 90 phosphorylation-sites, including 60 Ser redidue,18 Thr redidue and 12 Tyr redidue. SMART online analysis suggested that PHIP protein possess eight WD40 domains, one browodomain and five low complexity domains.(4) Comparing with control group,5 mmol/L glucose (low concentration)had tiny effects on promoting PHIP, IRS1 and TBC1D1 expression. Within 30 mmol/L glucose, the expression of PHIP, IRS1 and TBC1D1 were significantly increased (P<0.05).Low concentration of insulin could promote expression of PHIP, IRS1 and TBC1D1, while high concentration of insulin (200 nmol/L) could significantly decrease their expressions (P<0.05).(5) When synergistic the effect of insulin and glucose, low concentration of insulin (50 nmol/L) with 5 mmol/L and 30 mmol/L glucose treatment could increase the expression of PHIP, IRS1 and TBC1D1. However, the high concentration of insulin (100 nmol/L) with both of the low and high concentrations of glucose could inhibit their expressions.