色瓦绵羊RPL8基因的组织表达特征及生物信息学分析

Tissue expression characteristics and bioinformatics analysis of RPL8 gene in Sewa sheep

【目的】研究色瓦绵羊核糖体蛋白L8基因(RPL8)在各组织部位的表达特征及其潜在功能作用,揭示RPL8基因调控色瓦绵羊产奶质量的分子作用机制,为推进色瓦绵羊的分子遗传育种提供理论依据。【方法】PCR扩增色瓦绵羊RPL8基因,通过ProtParam、ProtScale、SignalP 5.0、TMHMM 2.0等在线软件分析RPL8基因结构特征及潜在功能,并以实时荧光定量PCR检测其在色瓦绵羊各组织中的表达分布情况。【结果】RPL8基因在色瓦绵羊乳腺、肌肉、心脏、肾脏、脾脏、肝脏、小肠、瘤胃和卵巢等9个组织中均有表达,在小肠、乳腺和卵巢中的相对表达量极显著高于其他组织中的相对表达量(P<0.01)。色瓦绵羊RPL8基因编码区(CDS)序列长774 bp,共编码257个氨基酸残基;基于RPL8基因核苷酸序列相似性构建的系统发育进化树显示,色瓦绵羊与山羊、牦牛、牛等反刍物种的亲缘关系较近。色瓦绵羊RPL8蛋白相对分子量为28024.66Da,理论等电点(pI)为11.04,是一种稳定的碱性亲水性蛋白,不具有跨膜结构,主要定位于细胞质(占78.3%);色瓦绵羊RPL8蛋白存在15个磷酸化位点[7个丝氨酸(S)位点,8个苏氨酸(T)位点],其二、三级结构中均以无规则卷曲为主。色瓦绵羊RPL8蛋白的互作蛋白均为核糖体蛋白,其同源性高达99.90%,其中,RPS5、RPS16、RPL11、RPS3、RPS18和RPS11属于通用核糖体蛋白家族,RPL13属于真核核糖体蛋白家族。【结论】色瓦绵羊RPL8是一种胞内稳定的碱性亲水性蛋白,具有核糖体蛋白的基本结构特征。RPL8基因在色瓦绵羊各组织中的表达具有普遍性,尤其在乳腺和卵巢中呈高表达,说明RPL8基因除了发挥其家族基本功能作用外,在色瓦绵羊乳腺和卵巢组织中可能还影响乳脂含量与卵泡的周期性发育。

【Objective】To study the expression and potential functional role of ribosomal protein L8 gene(RPL8)in different tissues of Sewa sheep,and to reveal the molecular mechanism of RPL8 gene in regulating milk quality of Sewa sheep,so as to provide theoretical basis for promoting molecular genetic breeding of Sewa sheep.【Method】The RPL8 gene was amplified by PCR and its structure and function were analyzed by ProtParam,ProtScale,SignalP 5.0 and TMHMM 2.0 softwares.Real-time fluorescence quantitative PCR was used to detect its expression and distribution in va-rious tissues of Sewa sheep.【Result】RPL8 gene was expressed in nine tissues of Sewa sheep,including mammary gland,muscle,heart,kidney,spleen,liver,small intestine,rumen and ovary,and the relative expression levels in small intes‐tine,mammary gland and ovary were extremely significantly higher than those in other tissues(P<0.01).The coding re‐gion(CDS)of RPL8 gene in Sewa sheep was 774 bp in length,encoding 257 amino acid residues.The phylogenetic tree constructed based on the nucleotide sequence similarity of RPL8 gene showed that Sewa sheep was closely related to goat,yak,cattle and other ruminant species.The relative molecular weight of RPL8 protein was 28024.66 Da,and the theoretical isoelectric point(pI)was 11.04.RPL8 protein was a stable basic hydrophilic protein,without transmembrane structure,and mainly located in the cytoplasm(78.3%).There were 15 phosphorylation sites[7 serine(S)sites,8 threo‐nine(T)sites]in the RPL8 protein of Sewa sheep.The secondary and tertiary structure of RPL8 protein were mainly ran‐dom coil.The interaction proteins of RPL8 protein from Sewa sheep were all ribosomal proteins,and the homology was as high as 99.90%.Among them,RPS5,RPS16,RPL11,RPS3,RPS18 and RPS11 belonged to the universal ribosomal protein family,and RPL13 belonged to the eukaryotic ribosomal protein family.【Conclusion】Sewa sheep RPL8 is an in‐tracellular stable,basic,hydrophilic protein that has the basic structural features of a ribosomal protein.The expression of RPL8 gene is universal in all tissues of Sewa sheep,especially in the mammary gland and ovary,which indicates that RPL8 gene may not only play a basic role in its family function,but also affect milk fat content and periodic development of follicles in the mammary gland and ovary of Sewa sheep.