小尾寒羊脂联素基因的克隆测序、结构特征与表达分析

发布时间：2018-03-05 12:39

本文选题：小尾寒羊　切入点：脂联素基因　出处：《山东农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：脂肪对于绵羊维持正常生命活动及生理代谢发挥重要的作用,脂肪性状是影响绵羊肌肉品质的一个重要的经济性状。脂联素(Adipoq)是脂肪组织分泌的一种内源性特异蛋白质,也是一种重要的脂肪细胞因子。本试验克隆了小尾寒羊脂联素基因的全长cDNA序列,并对该基因及其所编码的蛋白、mRNA水平和蛋白水平上的表达丰度进行了分析。主要研究结果如下:1.采用RT-PCR和RACE方法,以小尾寒羊组织为试验材料,克隆了小尾寒羊脂联素基因的全长cDNA序列,获得GenBank登录号为KM216385。该基因cDNA全长为2616 bp,包含720 bp的开放阅读框、76 bp的5'非编码区(5'UTR)和1820 bp的3'非编码区(3'UTR)。其中,开放阅读框编码239个氨基酸。2.生物信息学分析显示,脂联素基因编码的蛋白分子量为26 kDa,等电点为5.88,并存在信号肽结构,能被分泌到细胞外发挥作用。在氨基酸序列中,发现2个苏氨酸、3个丝氨酸和3个酪氨酸残基存在磷酸化位点,并发现存在5个O-糖基化位点而无N-糖基化位点。与其他物种的氨基酸同源性分析,证明绵羊脂联素蛋白的氨基酸序列与山羊的相似性最高。3.检测到Adipoq基因在小尾寒羊和杜泊羊中呈现出组织表达差异性。不同组织间,脂联素基因在皮下脂肪、肾周脂肪、腹股沟脂肪、臂二头肌、心血管组织中有表达,在肝脏、肾脏、卵巢组织中未检测到表达,提示脂联素基因的表达有一定的组织特异性。其中,该基因在三种脂肪组织中表达水平最高,极显著高于其他组织(P0.01),但是三种脂肪组织之间的表达量无显著差异(P0.05)。不同品种间,脂联素基因在杜泊羊臂二头肌组织中的表达极显著高于小尾寒羊(P0.01)。4.检测到Adipoq蛋白在小尾寒羊和杜泊羊中存在组织表达差异性。不同组织间,脂联素蛋白在皮下脂肪、肾周脂肪、腹股沟脂肪、臂二头肌、心血管组织中有表达,在肝脏、肾脏组织中未检测到表达。其中,脂联素蛋白在三种脂肪组织中表达量最高,极显著高于臂二头肌和心血管(P0.01),但是三种脂肪组织之间的表达量无显著差异(P0.05);不同品种间,脂联素蛋白在三种脂肪组织中的表达量,小尾寒羊均高于杜泊羊,但差异均不显著(P0.05)。杜泊羊臂二头肌的脂联素蛋白表达量高于小尾寒羊,而心血管中脂联素蛋白表达水平低于小尾寒羊,但是差异也不显著(P0.05)。综上所述,本研究成功克隆了小尾寒羊脂联素基因,对该基因序列及其编码的氨基酸序列的结构特征进行了预测,并比较分析了小尾寒羊和杜泊羊脂联素基因mRNA水平和蛋白水平上的组织表达谱特征,为后续研究脂联素的功能提供了一定的依据。
[Abstract]:Fat plays an important role in maintaining normal life activity and physiological metabolism in sheep. Adiponectin (Adipoq) is an endogenous protein secreted by adipose tissue, which is an important economic trait affecting the muscle quality of sheep. In this study, we cloned the full length cDNA sequence of adiponectin gene from small tail Han sheep. The mRNA level and protein abundance of the gene were analyzed. The main results were as follows: 1. Using RT-PCR and RACE methods, the tissues of small tail Han sheep were used as experimental materials. The full-length cDNA sequence of adiponectin gene of small tail Han sheep was cloned and the GenBank accession number was KM216385.The cDNA length of the gene was 2616 BP, containing 720bp open reading frame 76 BP 5'noncoding region 5 'unencoded UTRR and 1820 BP 3'noncoding region 3' UTR. among them, Open reading frame encodes 239 amino acids. Bioinformatics analysis shows that adiponectin gene encodes protein with a molecular weight of 26 kDa and a isoelectric point of 5.88, and has a signal peptide structure that can be secreted to play an extracellular role. Two threonine, three serine and three tyrosine residues were found to have phosphorylation sites, and five O-glycosylation sites without N-glycosylation sites were found. The results showed that the amino acid sequence of sheep adiponectin had the highest similarity with goat. 3. The difference of tissue expression of Adipoq gene was found in small tail Han sheep and du Bo sheep. Adiponectin gene was found to be subcutaneous fat and perirenal fat in different tissues. The expression of adiponectin gene in inguinal fat, biceps arm muscle, cardiovascular tissue was not detected in liver, kidney and ovary tissues, suggesting that adiponectin gene expression has certain tissue specificity. The highest expression level of the gene was found in three kinds of adipose tissues, which was significantly higher than that in other tissues, but there was no significant difference in the expression levels among the three kinds of adipose tissues. The expression of adiponectin gene was significantly higher in the biceps of arm than that in the small tail Han sheep (P 0.01). 4. The difference of tissue expression of adiponectin was detected between small tail Han sheep and du Bo sheep. Adiponectin was found in subcutaneous fat and perirenal fat in different tissues. Inguinal fat, biceps arm muscle, cardiovascular tissue, no expression in liver and kidney tissue, adiponectin in three kinds of adipose tissue, adiponectin expression is the highest in the three kinds of adipose tissue, The expression of adiponectin in the three adipose tissues was significantly higher than that in the biceps arm muscle and the cardiovascular tissue, but there was no significant difference among the three adipose tissues, and the expression of adiponectin in the three kinds of adipose tissues was higher in small tail Han sheep than that in Dupo sheep. But the difference was not significant (P 0.05). The adiponectin expression in the biceps of arm was higher than that in the small tail Han sheep, but the level of adiponectin in cardiovascular was lower than that in the small tail Han sheep, but the difference was not significant (P 0.05). In this study, the adiponectin gene of small tail Han sheep was cloned successfully, and the structural characteristics of the gene and the amino acid sequence were predicted. The tissue expression profiles of adiponectin gene mRNA and protein in small tail Han sheep and Dupo sheep were compared and analyzed, which provided a basis for further study of adiponectin function.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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