藏猪与大约克夏猪Mx1基因CDS区多态性及生物信息学分析

Polymorphism and bioinformatics analysis of Mx1 gene CDS region in Tibetan pigs and Yorkshire pigs

为了解藏猪与大约克夏猪Mx1基因编码序列(coding sequence, CDS)多态性,试验选择健康的成年(180日龄以上)大约克夏猪20头和藏猪30头为试验动物,采用PCR法扩增两个猪群的Mx1基因CDS区,测序分析其单核苷酸多态性(single nucleotide polymorphisms, SNPs),采用生物信息学方法分析Mx1基因CDS区编码蛋白质的理化性质、疏水性、磷酸化位点、二级结构、三级结构和与其他蛋白质的相互作用,分析SNPs位点对Mx1基因编码蛋白的上述指标的影响。结果表明:在藏猪Mx1基因CDS区存在C867G、A923C、A1241G和C1324A 4个SNP位点,在大约克夏猪中存在A1241G和C1324A 2个SNP位点;其中C867G、A923C和A1241G位点属于错义突变,分别使第289位由天冬氨酸变为谷氨酸,第308位由谷氨酸变为丙氨酸,第414位由赖氨酸变为精氨酸;C1324A为同义突变。Mx1基因CDS区共编码663个氨基酸,编码蛋白质呈酸性、亲水性,二级结构以α螺旋为主;共有55个磷酸化位点,其中有37个丝氨酸(Ser)磷酸化位点,13个苏氨酸(Thr)磷酸化位点,5个酪氨酸(Tyr)磷酸化位点;与干扰素刺激基因15(interferon stimulated exonuclease gene15,ISG15)、泛素特异性肽酶18(ubiquitin specific peptidase 18,USP18)、DDX解旋酶58(DEAD-box helicase58,DDX58)、真核翻译起始因子2α激酶2(eukaryotic translation initiation factor 2 alpha kinase 2,EIF2AK2)、干扰素诱导的GTP结合蛋白2(myxovirus resistance dynamin like GTPase 2,Mx2)、结构域包含蛋白5(HECT and RLD domain containing E3 ubiquitin protein ligase 5,HERC5)、2′,5′-寡腺苷酸合成酶1(2′,5′-oligoadenylate synthetase 1,OAS1)、Janus激酶1(JAK1)、cub2结构域蛋白(IRG6)、OAS样蛋白(OASL)存在互作关系。Mx1基因CDS的SNPs导致的氨基酸变化只引起蛋白质的二级结构、疏水性和磷酸化位点发生改变,没有改变其三级结构。说明Mx1基因编码蛋白依旧发挥原始功能。

In order to explore the polymorphism of coding sequence(CDS) of Mx1 gene in Tibetan pigs and Yorkshire pigs, twenty healthy adult Yorkshire pigs(over 180 days of age) and 30 healthy adult Tibetan pigs(over 180 days of age) were selected as experimental animals. The CDS of Mx1 gene was amplified by PCR and single nucleotide polymorphisms(SNPs) were analyzed by sequencing.Bioinformatics methods were used to analyze the physicochemical properties, hydrophobicity, phosphorylation site, secondary structure, tertiary structure and interaction with other proteins of the CDS-encoded protein of Mx1 gene, and to analyze the effects of SNPs site on the above indicators of CDS-encoded protein of Mx1 gene. The results showed that four SNPs(C867 G, A923 C, A1241 G and C1324 A) were found in CDS region of Tibetan pig Mx1 gene, and two SNPs(A1241 G and C1324 A) were found in Yorkshire pigs.Among them, C867 G, A923 C and A1 241 G were missense mutations;the corresponding aspartic acid at position 289 becomes glutamic acid;glutamic acid at position 308 becomes alanine;and lysine at position 414 becomes arginine, while C1324 A was synonymous mutation.A total of 663 amino acids were encoded in the CDS of Mx1 gene, and the coding protein was acidic and hydrophilic, with α helix as the main secondary structure. There were 55 phosphorylation sites, including 37 serine(Ser) phosphorylation sites, 13 threonine(Thr) phosphorylation sites, and 5 tyrosine(Tyr) phosphorylation sites. The Mx1 gene encodes proteins with interfer on stimulated exonuclease gene15(ISG15), ubiquitin specific peptidase 18(USP18), DDX-box helicase58(DDX58), eukaryotic translation initiation factor 2 alpha kinase 2(EIF2 AK2), myxovirus resistance dynamin like GTPase 2(Mx2), structural domain containing protein 5(HECT and RLD domain containing E3 ubiquitin protein ligase 5, HERC5), 2′,5′-oligoadenylate synthetase 1(OAS1), Janus kinase 1( JAK1), cub2 structural domain protein(IRG6), and OAS-like protein(OASL) are in reciprocal relationship.In addition, the changes of amino acids caused by SNPs in CDS of Mx1 gene only affected the secondary structure, hydrophobicity and phosphorylation sites of the protein, they but did not change the properties of the tertiary structure. The results indicated that Mx1 protein still played its original function.