鸡TRAF6基因外显子区功能性SNP预测与分析

Prediction and Analysis of Functional SNP on Exon Region of Chicken TRAF6 Gene

采用生物信息学方法筛选鸡TRAF6基因中的功能性非同义单核苷酸多态性(nsSNPs)位点。从dbSNP数据库中检索出TRAF6基因的8个nsSNPs,利用生物信息学软件PROVEN、SIFT、PhD-SNP和SNAP2分别对nsSNPs进行功能性预测;使用I-Mutant2.0和Mupro对突变位点氨基酸的稳定性进行预测;此外,利用Clustal Omega和Jalview对鸡TRAF6基因编码的氨基酸序列进行多序列比对和进化位点保守性预测。使用MutPred2预测突变可能造成的影响;最后使用Sopma和SWISS-MODEL软件分别预测TRAF6野生型和突变型的蛋白质二级结构和构建它们的三级结构。结果表明:从TRAF6基因的nsSNPs位点筛选出来3个有害突变位点rs736890315(S17R)、rs734934585(C20G)和rs734774178(V472E),其中V472E是四种软件共同筛选出的突变位点。V472E突变位点可能影响鸡TRAF6蛋白的功能,且所有位点突变都会使TRAF6蛋白稳定性降低。保守性分析显示,D16G、S17R和A307T为不保守位点,C20G、S40G、S40R、T42A和V472E为保守的功能性残基;MutPred2预测结果发现,V472E位点的突变导致蛋白质的稳定性改变,其他位点的突变均无显著影响;二级结构分析结果表明,鸡TRAF6蛋白主要以无规则卷曲和α螺旋为主,V472E、S40G和C20G这三个位点上的突变都导致了无规则卷曲百分比的提高和α螺旋百分比下降;三级结构分析结果发现,TRAF6蛋白的野生型和突变体的三级结构与二级结构预测结果一致。本研究预测发现V472E位点突变可能严重影响鸡TRAF6蛋白质的结构,V472E可能是鸡TRAF6基因的潜在功能性位点。

The purpose of this study was to screen the gene of chicken TRAF6 functional non-synonymous single nucleotide polymorphisms(nsSNPs)by the bioinformatics method.In this study,the 8 nsSNPs of TRAF6 gene were retrieved from dbSNP database,and the functional prediction of nsSNPs was performed by using bioinformatics software PROVEN,SIFT,PhD-SNP,and SNAP2,respectively;The amino acid stability of mutation sites was analyzed by I-Mutant2.0 and Mupro methods;In addition,Clustal Omega and Jalview were used to perform multiple sequence alignment and conservatively predict the evolution sites of amino acid sequences encoded by chicken TRAF6 gene,and Mutpred2 was used to predict the possible effects of mutations.Finally,Sopma and SWISS-MODEL software were used to predict the secondary structure and construct the tertiary structure of TRAF6 wild-type and mutant proteins,respectively.The results showed that three harmful mutation sites,rs736890315(S17 R),rs734934585(C20 G),and rs734774178(V472 E),were screened out from nsSNPs sites of TRAF6 gene,of which V472 E was the sites jointly screened by the four software.The V472 E mutation site might affect the function of chicken TRAF6 protein and all site mutations would reduce the stability of TRAF6 protein.Conservative analysis showed that D16 G,S17 R,and A307 T were not conserved sites,and C20 G,S40 G,S40 R,T42 A,and V472 E were conserved functional residues;MutPred2 prediction results revealed that V472 E mutation site led to changes in protein stability,while mutation at all other sites had no significant effects.The results of secondary structure analysis showed that the chicken TRAF6 protein was composed of the irregular coil and alpha helix.The mutations at V472 E,S40 G,and C20 G all led to the decrease of the irregular coil and the increase of alpha helix.The tertiary structure analysis showed that the tertiary structure of the wild-type and mutant of TRAF6 protein were consistent with the predicted secondary structure.This study predicted that V472 E mutations might seriously affect the structure of chicken TRAF6 protein,and V472 E may be a potential functional site of the chicken TRAF6 gene.