皱皮香猪HAS 2基因SNPs的鉴定及生物信息学分析

SNPs Identification and Bioinformatics Analysis of the Hyaluronan Synthase 2 Gene in Xiang Pig with Wrinkled Skin

为了探究皱皮香猪全身性皮肤褶皱是否与透明质酸合成酶2(hyaluronan synthase 2,HAS 2)基因变异有关,本研究以普通香猪为对照,采用特异性PCR方法克隆HAS 2基因的外显子编码区,应用生物信息学方法分析测定基因编码区的碱基变异,检测变异位点在群体中的分布频率。结果显示,皱皮香猪HAS 2基因中检测到4个SNPs位点:位于外显子1的T221A错义突变(导致亮氨酸替换为赖氨酸)和A228G同义突变,位于外显子2的T183C和外显子4的C537T同义突变。生物信息学分析发现,T221A和A228G位点构成4种单倍型,其中T-A为皱皮香猪群体的主要单倍型(27.5%),且在皱皮香猪群体中紧密连锁(r^(2)=0.253),而且T221A和A228G突变可引起mRNA自由能和蛋白质结构发生变化,TA组合的自由能为-573.29 kJ·mol^(-1),TG组合的自由能为-580.89 kJ·mol^(-1),AG组合的自由能为-572.66 kJ·mol^(-1);AA组合的自由能为-571.03 kJ·mol^(-1);蛋白序列中亮氨酸替换为赖氨酸后,α螺旋由35.52%降为32.97%,自由卷曲由43.17%升至44.51%,同时引起蛋白序列三级结构的改变。位点T221A和A228G在香猪群体中呈现出丰富的多态性,均表现出3种基因型;关联分析结果显示,皱皮香猪T221A位点的A等位基因频率显著高于普通香猪(P<0.05),A228G位点中皱皮香猪的G等位基因频率极显著高于普通香猪(P<0.01)。T183C位点的C等位基因频率和C537T位点的T等位基因频率在皱皮香猪和普通香猪间未达到显著性差异(P>0.05)。研究结果揭示,HAS 2基因的外显子1中发生的两个碱基变异可能影响基因转录后mRNA的稳定性以及蛋白的三维结构,影响HA的合成量,并与皱皮香猪体侧皮肤不均匀增厚且发生褶皱有关。

To explore whether the systemic skin wrinkle of Xiang pig with wrinkled skin(XPZ)was related to the variation of HAS 2(hyaluronan synthase 2)gene,all exon regions of HAS 2 gene were cloned by specific PCR method with ordinary Xiang pig(XP)as control.The base variation of gene coding region was analyzed by bioinformatics method,and the distribution frequencies of variant SNP sites in population were detected.Four SNP sites were detected from HAS 2 gene of Xiang pig with wrinkled skin,including T221A missense mutation in exon 1(resulting in leucine substitution to lysine)and A228G synonymous mutation,T183C synonymous mutation in exon 2 and C537T synonymous mutation in exon 4.Bioinformatics analysis showed that 4 haplotypes were formed at T221A and A228G loci.Among them,T-A was the main haplotype(27.5%)and closely linked(r^(2)=0.253)in Xiang pig population with wrinkled skin.Moreover,mutations in T221A and A228G could change HAS 2 mRNA free energy and protein structure,and the free energy of TA,TG,AG and AA combinations were-573.29,-580.89,-572.66,-571.03 kJ·mol^(-1),respectively.After leucine was replaced by lysine resulted from T221A missense mutation,the percentage ofα-helix decreased from 35.52%to 32.97%,and the free crimp increased from 43.17%to 44.51%.Two SNPsites of loci T221A and A228G showed abundant polymorphism in Xiang pig population with 3 genotypes.And both frequencies of A allele in T221A(P<0.05)and G allele in A228G(P<0.01)were significantly higher in Xiang pig with wrinkled skin than that in common Xiang pig.There were no significant difference in the frequencies of C allele in T183C and T allele in C537T between Xiang pig with wrinkled skin and common Xiang pig.The results showed that the two base mutations in exon 1 of HAS 2 gene might affect the stability of post-transcriptional mRNA,the three-dimensional structure of protein and HA synthesis,and have a relationship to the thickening and wrinkling of skin in Xiang pig with wrinkled skin.