USP26基因在热应激前后绵羊睾丸中的差异表达及生物信息学分析

Differential Expression and Bioinformatics Analysis of USP26 Gene in Sheep Testis before and after Heat Stress

试验旨在探究USP26基因在热应激前后的吐鲁番黑羊及萨福克羊睾丸中的差异表达,以期为研究USP26的生物学功能及作用机制提供依据。通过生物信息学软件分析绵羊USP26基因序列、编码蛋白的理化性质、疏水性、跨膜区域、信号肽、二级结构和三级结构,然后选取热应激前后的吐鲁番黑羊及萨福克羊活体去势后提取睾丸总RNA,反转录后进行实时荧光定量PCR,分析USP26基因在热应激前后不同羊之间mRNA的差异表达。结果表明,绵羊USP26基因序列与山羊的亲缘关系最近,与弯角大羚羊次之,与野猪最远。绵羊USP26蛋白分子量100617.20,不稳定系数46.91,总平均亲水系数-0.580,属于不稳定的亲水性蛋白;通过对蛋白亚细胞定位发现,绵羊USP26蛋白主要存在于细胞核(69.6%)和细胞质(17.4%)中;绵羊USP26蛋白不属于跨膜蛋白和分泌蛋白;通过对二级结构和三级结构预测发现,其主要包含α-螺旋、β-转角、延伸链和无规则卷曲4种结构,其中α-螺旋(39.27%)及无规则卷曲(44.75%)占绝大部分。吐鲁番黑羊的USP26基因在热应激前后的睾丸中mRNA的表达无差异(P>0.05),但萨福克羊的USP26基因在热应激前后的睾丸中mRNA的表达差异显著(P<0.05)。综上所述,绵羊USP26基因与山羊进化关系较近,USP26蛋白为无跨膜结构、无信号肽的不稳定亲水性蛋白;USP26基因在热应激前后的吐鲁番黑羊中表达无差异,但在萨福克羊中显著降低,表明热应激对USP26基因的表达有影响,进一步推测热应激后萨福克羊USP26基因表达的下降可能会导致雄激素的下降及精液品质的降低。

This experiment was conducted to explore the differential expression of USP26 gene in Turpan Black sheep and Suffolk sheep before and after heat stress,and analyze the bioinformatics of USP26 gene.The sequence,physicochemical properties,hydrophobicity,transmembrane region,signal peptide,secondary structure and tertiary structure of sheep USP26 gene were analyzed by bioinformatics software.Then,the total RNA was extracted from the castrated Turpan Black sheep and Suffolk sheep before and after heat stress,and the real-time fluorescent quantitative PCR was performed after reverse transcription to analyze the differential expression of USP26 mRNA in different sheep before and after heat stress.Phylogenetic tree analysis showed that sheep USP26 gene sequence had the closest genetic relationship with goat,followed by horned antelope and wild boar.The molecular weight of sheep USP26 protein was 100617.20,the instability coefficient was 46.91,and the total average hydrophilic coefficient was-0.580,which belonged to unstable hydrophilic protein.The subcellular localization of protein revealed that sheep USP26 protein mainly existed in nucleus(69.6%)and cytoplasm(17.4%).The results of transmembrane region and signal peptide prediction showed that sheep USP26 protein did not belong to transmembrane protein and secretory protein.Secondary structure and tertiary structure prediction found that it mainly contained four kinds of structures:α-helix,β-turn,extended chain and random coil,of whichα-helix(39.27%)and random coil(44.75%)accounted for the majority.The results of real-time fluorescent quantitative PCR showed that there was no difference in the mRNA expression of USP26 gene in the testis of Turpan Black sheep before and after heat stress(P>0.05),but the mRNA expression of USP26 gene in the testis of Suffolk sheep before and after heat stress was significantly different(P<0.05).In summary,the sheep USP26 gene has a close evolutionary relationship with goats,and the USP26 protein is an unstable hydrophilic protein without transmembrane structure and signal peptide.There was no difference in the expression of USP26 gene in Turpan Black sheep before and after heat stress,but it was significantly decreased in Suffolk sheep,indicating that heat stress had an effect on the expression of USP26 gene.It was further speculated that the decrease of USP26 gene in Suffolk sheep after heat stress might lead to the decrease of androgen and the decrease of semen varieties.