色瓦绵羊GPIHBP1基因克隆及其组织表达特征分析

Cloning and tissue expression characteristics analysis of GPIHBP1 gene in Sewa sheep

  • 摘要: 【目的】克隆色瓦绵羊乳腺组织糖基化磷脂酰肌醇锚定高密度脂蛋白结合蛋白1基因(GPIHBP1),明确其生物信息学特性及组织表达分布特征,为揭示GPIHBP1基因影响绵羊产奶性状的作用机制提供参考依据。【方法】克隆色瓦绵羊GPIHBP1基因编码区(CDS)序列,通过Prot Para、Prot Scale、SOPMA、SWISS-MODEL、TMHMM-2.0等在线软件进行生物信息学分析,并以实时荧光定量PCR检测该基因在色瓦绵羊乳腺、肌肉、心脏、肾脏、肝脏、小肠、瘤胃和卵巢等8个组织中的表达情况。【结果】色瓦绵羊GPIHBP1基因CDS序列长519 bp,共编码172个氨基酸残基,与山羊GPIHBP1氨基酸序列(XM_018058666.1)比对,有4处氨基酸发生突变(32A→P、52V→A、130S→N和170M→V);色瓦绵羊GPIHBP1蛋白分子量为18063.87 Da,理论等电点(p I)为4.29,不存在跨膜螺旋结构,但存在信号肽(位于第20~21位氨基酸处),是一种细胞外的酸性亲水性蛋白。色瓦绵羊GPIHBP1蛋白存在12个磷酸化位点(6个丝氨酸磷酸化位点,5个苏氨酸磷酸化位点,1个酪氨酸磷酸化位点),其二级结构以无规则卷曲状态(占55.07%)为主,其次是α-螺旋(占35.47%),延伸链、β-转角分别占5.81%和4.65%。GPIHBP1基因在色瓦绵羊乳腺、肌肉、心脏、肾脏、肝脏、小肠、瘤胃和卵巢等8个组织中均有表达,以乳腺中的相对表达量最高,其次是心脏和肝脏,均极显著高于肌肉中的相对表达量(P<0.01)。【结论】GPIHBP1基因在色瓦绵羊不同组织中均有表达,且以乳腺中的相对表达量最高,可能与乳脂相关基因表达的增减有关,其中,GPIHBP1基因与LPL基因共同作用而参与绵羊泌乳调控。

     

    Abstract: 【Objective】The glycosylated phosphatidylinositol anchored HDL-binding protein 1 gene (GPIHBP1) in the mammary gland tissue of Sewa sheep was cloned to clarify its bioinformatics characteristics and tissue expression dis-tribution characteristics,which provided a reference for revealing the mechanism of GPIHBP1 genes affecting milk pro-duction traits in sheep.【Method】The sequence of coding region (CDS) of GPIHBP1 gene of Sewa sheep was cloned,and bioinformatics analysis was carried out by online softwares such as Prot Para,Prot Scale,SOPMA,SWISS-MODEL,and TMHMM-2.0,and real-time fluorescence quantitative PCR was used to detect the expression of this gene in mam-mary gland,muscle,heart,kidney,liver,small intestine,rumen and ovary in sewa sheep.【Result】The CDS sequence of Sewa sheep GPIHBP1 gene was 519 bp long,encoding a total of 172 amino acid residues,and compared with the amino acid sequence of goat GPIHBP1 (XM_018058666.1),there were four amino acid changes(32A→P,52V→A,130S→N and 170M→V);the molecular weight of Sewa sheep GPIHBP1 protein was 18063.87 Da,and the theoretical isoelectric point (p I) was 4.29.There was no transmembrane helical structure,but there was a signal peptide (located at amino acids20-21),which was an extracellular acidic hydrophilic protein.There were 12 phosphorylation sites (6 serine phosphoryla-tion sites,5 threonine phosphorylation sites,1 tyrosine phosphorylation site) existed in the Sewa sheep GPIHBP1 pro-tein,and its secondary structure was in the state of irregularly curls (55.07%),followed by α-helix (35.47%),and the extended strand and β-turns accounted for 5.81%and 4.65%,respectively.GPIHBP1 gene was expressed in eight tissues including mammary gland,muscle,heart,kidney,liver,small intestine,rumen and ovary of Sewa sheep,and the highest relative expression was found in the mammary gland,followed by the heart and liver,which were both extremely signifi-cant higher than that in the muscle (P<0.01).【Conclusion】GPIHBP1 gene is expressed in different tissues of Sewa sheep,and the relative expression in mammary gland is the highest,which might be related to the increase or decrease in the expression of milk fat-related genes,among which,GPIHBP1 gene acts together with LPL gene and participate in the regulation of lactation in sheep

     

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