撒坝猪SPDEF基因克隆及其生物信息学分析

Cloning and Bioinformatics Analysis of SPDEF Gene in Saba Pigs

为明确猪SPDEF基因的遗传特性,试验对不同猪种SPDEF基因的CDS区进行序列扩增、测序、拼接,并对撒坝猪SPDEF基因CDS序列进行生物信息学分析。采用PCR直接测序法测定猪SPDEF基因外显子序列,运用SeqMan进行序列拼接。采用生物信息学软件分析撒坝猪SPDEF基因开放阅读框及其编码蛋白的基本理化性质、疏水性、信号肽、跨膜区、亚细胞定位、磷酸化位点、糖基化位点、二级结构、三级结构、功能结构域,并构建系统进化树。结果表明,各猪种SPDEF基因编码区仅在外显子1、2分别检测到1个同义突变(C13971T、C16541T),表明该基因编码区在所检测的猪群中高度保守。生物信息学分析发现,该基因有1个长1 092 bp的完整开放阅读框;SPDEF蛋白中丝氨酸(Ser)含量最高(10.7%),体外理论半衰期为30 h,属于不稳定的亲水性蛋白;该蛋白不存在信号肽及跨膜结构,存在39个潜在的磷酸化位点和1个糖基化位点,亚细胞定位于细胞质;二级结构预测中,参与无规则卷曲的氨基酸最多(55.10%),其次是α-螺旋(28.37%);功能结构域预测发现,该蛋白存在1个ETS功能结构域和1个SAM-PNT功能结构域。系统进化树结果显示,撒坝猪与牛、绵羊和山羊的亲缘关系较近。本研究为进一步分析撒坝猪SPDEF基因的功能奠定了一定的理论基础。

In order to identify the genetic characteristics of the SPDEF gene,the CDS of different pig breeds were amplified,sequenced,spliced,and the CDS of Saba pigs were analyzed by bioinformatics.The exon sequence of pigs were detected by direct sequencing after PCR,and sequence splicing were performed using SeqMan.The open reading frame(ORF) of SPDEF gene,and basic physicochemical properties,hydrophobicity,signal peptide,transmembrane region,subcellular localization,phosphorylation site,glycosylation site,secondary structure,tertiary structure,functional domain of SPDEF protein were predicted,and phylogenetic tree was constructed using bioinformatics software.The results showed that only 2 synonymous SNPs were detected in exon 1(C13971 T) and exon 2(C16541 T) of SPDEF gene in different pigs,respectively,indicating that the coding region of SPDEF gene was highly conserved among the tested herds.SPDEF gene had a complete ORF at length of 1 092 bp.The Ser content was the highest,which accounted for 10.7%.SPDEF was an unstable hydrophilic protein with an estimated half-life of 30 h in vitro.The SPDEF protein has no signal peptide and transmembrane structure,and had 39 potential phosphorylation sites and 1 glycosylation site.The protein located the cytoplasm in analysis of subcellular localization.In the secondary structure prediction,the amino acids involved in the random coil were the most,accounting for 55.10%,followed by the α-helix region,accounting for 28.37%.Functional domain predictions revealed the existence of an ETS functional domain and an SAM-PNT functional domain.Sequence homology analysis showed that pig has higher homology with Bos taurus,Ovis aries and Capra hircus.This study could provide references for further analysis of the function of SPDEF gene in Saba pigs.