Cloning And Expression Of Porcine CA? CDS And Its Effect On PK5 Cell Proliferation

Carbonic anhydrases,the reversible hydration reaction of CO2 catalyzed by a two-step reaction(CO2 + H2 O ? H2CO3 ?HCO3-+ H+)aim to form bicarbonate and protons to maintain the intracellular and extracellular acid-base balance.Carbonic anhydrase ? belongs to the CA family,but is different from other isoenzymes in tissue distribution and catalytic CO2 activity.The main causes are the change of amino acid at position 64 of the CA? protein(His?Lys)and two cysteines and two reactive sulfhydryl groups.At present,the CA? gene is mainly studied in muscle and fat phenotypes of human and mouse,while the phenotypic study on cell proliferation in pigs is relatively rare.Therefore,this test obtain the CDS sequence of porcine pigs CA? by RT-PCR and cloning sequencing.And the protein encoded by the gene was analyzed by bioinformatics.The expression of CA? m RNA in different tissues of Mashen pigs and muscular tissue of Large White pig and Mashen pig at different developmental stages was detected by q RTPCR.The pc DNA 3.1-EGFP-CA? vector by homologous recombination and three p HS-CR054-EGFPCA? vectors by CRISPR/Cas9 technology are transfected into PK15 cells.Then detecting the expression of ki67,Caspase3 and Parp2 m RNA by RT-PCR and the cell proliferation rate by CCK-8.Finally,20 ?mol/L methylation inhibitor was added to PK15 cells for 12 h to investigate whether the CA? gene was regulated by DNA methylation.The test results show that CA? encodes 260 amino acids.And the ?-helix and the ?-turn are folded to form a hydrophilic basic protein that functions as a signal-free peptide in the cytoplasm.CA? gene is expressed in various tissues.Large white pigs Large White pig and Mashen pig were lowly expressed at 0,1 and 2 months of age,and the difference was not significant.But the expression was increase at 3 months of age,and the difference was extremely significant.At 4 months of age,the expression began to decrease,and at 5 months of age Mashen pig it began to rise to highest expression level.Functional verification showed that the expression of ki67 m RNA was increased,the expression of apoptosis genes Caspare3 and Parp2 m RNA were decreased,and the cell proliferation rate was increased after the pc DNA 3.1-EGFP-CA? vector was transfected into PK15 cells.And the result of p HS-CR054-CA? test was reversed.The results of methylation assay showed that the addition of 20 ?mol/L methylation inhibitor increased the expression of CA? m RNA,and decreased the proliferation rate of PK15 cells.In summary,this study successfully cloned the CA? CDS region in pig and analyzed the bioinformatics characteristics and spatiotemporal representation of the gene.Successfully constructed overexpression and knockout vectors to investigate the effect of CA? gene on cell proliferation and to verify the regulation of CA? gene by methylation.The gene may be regulated by DNA methylation and thus affect the cell proliferation.