Isolation, Localization, Expression Profile And Polymorphism Detection Of Seven Pig Placenta Permeability Related Genes

The pig placenta is responsible for nutrient transport from the sow to the fetus during pregnancy. The capability of placenta nutrient transport depends on the density and permeability of blood vessels on the placental surface besides placenta size. A large number of potentially important growth factors may directly or indirectly regulate angiogenesis at the maternal fetal interface. Vascular endothelial growth factor (VEGF) is a potent angiogenic, vasoactive, permeability-increasing molecule. VEGF binding sites were identified in vascular endothelial cells corresponding to VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR). VEGF up-regulation and VEGFR activation promote numerous downstream targets of receptor tyrosine kinases of signal transduction pathways such as endothelial nitric oxide synthase (eNOS) and Vascular endothelial cadherin (CDH5), which lead to vascular endothelia cells proliferation and vascular permeability. This study was designed to clone the cDNA of pig VEGF, VEGFR1, VEGFR2, eNOS, iNOS, CDH5 and ARRB2 genes based on the sequences of human, rat, mouse and cattle genes by comparative genomies and bioinformatics approaches, to investigate the expression charaeteristic of these genes by RT-PCR; to detect the polymorphisms of iNOS gene by DNA sequeneing and PCR-RFLP methods. The main results are as follows:1. Partial cDNA sequences of pig VEGF, VEGFR1, VEGFR2, eNOS, iNOS, CDH5 and ARRB2 genes were cloned by homologous cloning and in silico cloning methods. We compared the amino acid sequences of these genes, with those of these genes from other species retrieved from the EMBL and GenBank databases. Phylogenic trees of VEGF, VEGFR1, VEGFR2, eNOS, CDH5 and ARRB2 based on the amino acid sequences were generated using the DNAMAN program.2. Primers were designed according to the sequences of these introns to map VEGF, VEGFR1 and VEGFR2 genes using radiation hybrid (RH) technique. These genes were mapped on 7q11-q12, 11 p13 and 8p12. The Positions of these genes are corresponding to those in human.3. The tissue expression levels of VEGF, VEGFR1, VEGFR2, eNOS, iNOS, CDH5 and ARRB2 genes were investigated by RT-PCR. Porcine VEGF, VEGFR1, VEGFR2, eNOS, iNOS, CDH5 and ARRB2 genes showed expression in all seven tissues detected: heart, liver, spleen, lung, kidney, skeletal muscle, and placenta.4. SYBR Green I-based real-time quantitative RT-PCR results demonstrated that from gestation day 33 to day 90, Landrace exhibited significant increase in placental VEGF and ARRB2 mRNA expression (p<0.05). Moreover, expression levels of VEGF, VEGFR1, VEGFR2, and eNOS mRNA was higher in the placenta of Erhualian than those in Landrace on Day 90 of gestation (p<0.01). Erhualian placental CDH5 and ARRB2 expression levels were significantly lower than those in Landrace conceptuses on day 90 of gestation (p<0.01).5. We found an A/C SNP in porcine iNOS gene CDS, developed PCR-Bsh1236 I-RFLP to detect this SNP. The SNP was genotyped by the PCR-Bsh1236 I-RFLP in a Large White population. Association analysis found that this SNP was significantly associated ADG100 (p<0.05).