Prediction And Analysis Of NsSNPs In The Exon Of Chicken BRD2 Gene And Study On Interaction And Expression Characteristics Of BRD2/TBP/E2F-1

Bromodomain-containing protein 2(BRD2)is a member of the BET protein family,which plays an important role in gene transcription regulation,cell proliferation and cellular immune regulation.TATA-binding protein(TBP)is a core DNA-binding subunit of the eukaryotic transcription factor TFIID,which can regulate the formation of transcription complexes and the binding ability of DNA to its own carboxyl terminus.Transcription factor E2F-1 acts as a DNA repair factor and plays a role in cellular DNA repair,cell proliferation and aptosis.At present,studies have shown that BRD2 gene is associated with the production traits of animals.Meanwhile,BRD2/TBP/E2F-1 interactions have only been reported in human and mouse.However,the amino acid homology of BRD2,TBP and E2F1 proteins among chicken and human,rat and mouse was low,and whether this will affect the inetarctions of chicken BRD2/TBP/E2F-1 remianed unknown.In addition,the expression characteristics of BRD2,TBP and E2F-1 genes in different tissues during chicken development had not been reported.Therefore,in this study,the non-synonymous single nucleotide polymorphisms(nsSNPs)in the coding region of chicken BRD2 gene were first analyzed to study its effect on BRD2 structure.The recombinant eukaryotic expression vectors of chicken BRD2,TBP and E2F-1 genes were then constructed to investigate the interactions among chicken BRD2,TBP,and E2F-1 proteins by fluorescence co-localization and co-immunoprecipitation assays.Moreover,real-time fluorescence quantitative PCR method was performed to detect the expression levels of BRD2,TBP and E2F-1 genes in the different tissues at different stages of chicken development.The main results are as follows:1.The presence of 32 nsSNPs in the chicken BRD2 gene was obtained from the db SNP database.Six common functional nsSNPs loci(rs740038469,rs738656993,rs732968462,rs740433852,rs739975970,and rs738254732)were screened by the bioinformatics software,including SIFT,PROVEAN,Ph D-SNP and Poly Phen-2.The results of multi-sequence alignment,the conservatism of polymorphism sites and the predictive analysis of protein stability showed that the T673P(rs738656993),L628W(rs732968462),M400R(rs740433852)and F98S(rs738254732)loci of chicken BRD2 protein were the conserved polymorphism site,and these mutations changed the stability of BRD2 protein,and altered the ratio of α helix,elongation chain,β rotation and random curl Therefore,the four mutated sites T673 P,L628W,M400 R and F98 S might be the main functional nsSNPs affecting the function of chicken BRD2 protein.2.The CDS regions of chicken E2F-1 and TBP genes were successfully amplified from the cDNA of chicken embryonic fibroblast cells.Meanwhile,the recombinant eukaryotic expression vectors p CMV-HA-E2F-1 and p CMV-Myc-TBP were correctly constructed.Bioinformatics analysis showed that the CDS regions of chicken E2F-1 and TBP genes were 1212 bp and 928 bp,respectively,encoding 403 and 302 amino acids.The molecular weight of chicken E2F-1 protein was about 43.5 k Da,and the secondary structure was mainly composed of irregularly coiled(54.09%)and α-helix(28.29%);the winged-helix DNA-binding structural domain of E2F-1protein was more conserved in chicken and other avina species than that in human and mammals,and the Dimerization domain of chicken E2F-1 protein existed multiple mutations.The molecular weight of chicken TBP protein was about 34.1 k Da,and its secondary structure mainly contained irregularly coiled(49.34%)and α-helical(27.48%);the PLN00062 domain of TBP protein was highly conserved in chicken,human and mammals.In addition,the amino acid homology of TBP and E2F-1 protein among chicken and human,rat and mouse was 58.6%～59.8% and96.4%～97.4%,respectively.Further fluorescence co-localization and co-immunoprecipitation assays suggested that chicken BRD2,TBP and E2F-1 proteins had obvious co-localization in the nucleus,and the three proteins had interactions.3.Real-time fluorescence quantitative PCR was used to detect the expression of BRD2,E2F-1and TBP genes in different tissues(eye,brain,heart,liver,lung,myogastric,pectoral and leg muscles)of Guizhou yellow chickens at different developmental stages [embryonic stage 14 d(E14d),1 day old(1d),7 days old(7d)and 14 days old(14d)].The results showed that chicken BRD2,TBP and E2F-1 genes were expressed in different tissues of Guizhou yellow chickens at all four stages.From E14 d to 14 d,the tissue with the highest expression of BRD2 gene at each stage was in the heart,while the lowest expression was in the lung;TBP gene had the highest expression in the lung and lowest in the leg muscle;E2F-1 gene was highest expressed in the liver and lowest expressed in the leg muscle.From E14 d to 1d,the expression of BRD2 and TBP genes in liver was stable,and the expression of BRD2 and TBP genes in gizzard increased;TBP and E2F-1 gene expression was stable in gizzard from 1d to 7d.Expression of BRD2,TBP and E2F-1 genes increased in the brain,but decreased in the pectoral muscle.From 7d to 14 d,BRD2 expression increased and TBP and E2F-1 gene expression decreased in gizzard.The above findings indicated that mutations at T673 P,L628W,M400 R and F98 S in nsSNPs of chicken BRD2 gene exon could affect the structure of BRD2 protein.Chicken BRD2,TBP and E2F-1 proteins interacted with each other in the nucleus.The chicken BRD2,TBP and E2F-1genes were expressed in different tissues at different stages of chicken embryo development,but the expression characteristics and expression trends of the three genbes were different.