PK-15细胞源EDC3分子的克隆及生物信息学分析
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摘要
为了研究猪肾细胞(PK-15)中mRNA脱帽激活因子(EDC3)基因并分析其编码区序列特征,试验采用巢式PCR方法获得PK-15细胞源EDC3基因并进行克隆与测序,利用生物信息学相关在线软件检测PK-15细胞与其他宿主EDC3基因的同源性及构建系统进化树,并对该基因编码蛋白的跨膜结构、信号肽等分子特性进行分析。结果表明:猪EDC3基因全长为1 326 bp,编码440个氨基酸,比预测片段少201 bp;与预测野猪的核苷酸序列同源性达99.0%,氨基酸序列同源性达97.5%;该基因与野猪的亲缘关系最近,处于同一分支,其次是瘤牛、绵羊;EDC3基因编码蛋白的二级结构中无规则卷曲所占比例为59.09%,α-螺旋所占比例为20.68%,该结构蛋白无信号肽与跨膜结构。
In this study, the PK-15 cell source EDC3 gene was obtained and cloned and sequenced using the nest-PCR method to analyze the sequence characteristics of the EDC3 gene coding region in PK-15 cells. Using bioinformatics related online software to discuss the homology of PK-15 cells and other host EDC3 genes and build a phylogenetic tree, and analyzed the molecular characteristics of the transmembrane structure and signal peptides of the gene coding protein.The results showed that the total length of the pig EDC3 gene was 1 326 bp, encoding 440 amino acids, 201 bp less than the predicted fragment. The homology of nucleotide sequence and amino acid sequence with wild boar were 99.0% and 97.5%.The gene was closest to the wild boar and they were in the same branch, followed by tumors and sheep. Bioinformatics analysis showed that the proportion of irregular curling in secondary structure of EDC3 gene coding protein was 59.09%, and the proportion of α-helix was 20.68%. The protein had no signaling peptide and transmembrane structure.
In this study, the PK-15 cell source EDC3 gene was obtained and cloned and sequenced using the nest-PCR method to analyze the sequence characteristics of the EDC3 gene coding region in PK-15 cells. Using bioinformatics related online software to discuss the homology of PK-15 cells and other host EDC3 genes and build a phylogenetic tree, and analyzed the molecular characteristics of the transmembrane structure and signal peptides of the gene coding protein.The results showed that the total length of the pig EDC3 gene was 1 326 bp, encoding 440 amino acids, 201 bp less than the predicted fragment. The homology of nucleotide sequence and amino acid sequence with wild boar were 99.0% and 97.5%.The gene was closest to the wild boar and they were in the same branch, followed by tumors and sheep. Bioinformatics analysis showed that the proportion of irregular curling in secondary structure of EDC3 gene coding protein was 59.09%, and the proportion of α-helix was 20.68%. The protein had no signaling peptide and transmembrane structure.
引文
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[14] SHETH U,PARKER R.Decapping and decay of messenger RNA occur in cytoplasmic processing bodies[J].Science,2003,300(5620):805-808.
[15] ROTHER R P,FRANK M B,THOMAS P S.Purification,primary structure,bacterial expression and subcellular distribution of an oocyte-specific protein in xenopus[J].Eur J Biochem,1992,206(3):673-683.
[16] LING S H,DECKER C J,WALSH M A,et al.Crystal structure of human EDC3 and its functional implications[J].Mol Cell Biol,2008,28(19):5965-5976.
[2] KSHIRSAGAR M,PARKER R.Identification of EDC3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae[J].Genetics,2004,166(2):729-739.
[3] ANANTHARAMAN V,ARAVIND L.Novel conserved domans in proteins with predicted roles in eukaryotic cell-cycle regula-tion,decapping and RNA stbility[J].BMC Genomics,2004,5(1):45.
[4] CHAN E K,YAO B,FRITZLER M J.Reflections on ten years of history of,and future prospects for,GW182 and GW/P body research[J].Adv Exp Med Biol,2013,768:261-270.
[5] TRITSCHLER F,EULALIO A,HELMS S,et al.Similar modes of interaction Enable Traler hitch and EDC3 to associate with dcp1 and me31b im distinct protein complexes[J].Mol Cell Biol,2008,28(21):6695-6708.
[6] DONG S,LI C,ZENKLUSEN D,et al.YRA1 autoregulation requires nuclear export and cytoplasmic Edc3p-mediated degradation of its pre-mRNA [J].Mol Cell,2007,25(4):559-573.
[7] EULALIO A,REHWINKEL J,STRICKER M,et al.Target-specific requirements for enhancers of decapping in miRNA-mediated gene sliencing[J].Genes Dev,2007,21(20):2558-2570.
[8] NISSAN T,RAJYAGURU P,SHE M,et al.Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms[J].Mol Cell,2010,39(5):773-783.
[9] TRITSCHLER F,BRAUN J E,EULALIO A,et al.Structural basis for the mutually exclusive anchoring of P body components EDC3 and Tral to the DEAD box protein DDX6/Me31B[J].Mol Cell,2009,33(5):661-668.
[10] DECKER C J,TEIXEIRA D,PARKER R.Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae[J].J Cell Biol,2007,179(3):437-449.
[11] BADIS G,SAVEANU C,FROMONT-RACINE M,et al.Targeted mRNA degradation by deadnylation –independent decapping[J].Mol Cell,2004,15(1):5-15.
[12] SCHELLER U,PFISTERER K,UEBE S,et al.Integrative bioinformtics analysis characterizing the role of EDC3 in mRNA decay and its assocition to intellectual disability[J].BMC Med Genomics,2018,11(1):41.
[13] FROMM S A,TRUFFAULT V,KAMENZ J,et al.The structural basis of EDC3 and scd6-mediated activation of the dcp1:dcp2 mRNA decaping complex[J].EMBO J,2012,31(2):279-290.
[14] SHETH U,PARKER R.Decapping and decay of messenger RNA occur in cytoplasmic processing bodies[J].Science,2003,300(5620):805-808.
[15] ROTHER R P,FRANK M B,THOMAS P S.Purification,primary structure,bacterial expression and subcellular distribution of an oocyte-specific protein in xenopus[J].Eur J Biochem,1992,206(3):673-683.
[16] LING S H,DECKER C J,WALSH M A,et al.Crystal structure of human EDC3 and its functional implications[J].Mol Cell Biol,2008,28(19):5965-5976.