摘要
【目的】克隆绵羊UCP4基因的编码序列(CDS),分析CDS及其编码蛋白结构特点,并探讨其mRNA的发育性表达规律,以期为该基因的结构和功能研究奠定理论基础。【方法】利用RT-PCR技术从绵羊大脑组织中扩增出该基因的编码区序列,运用生物信息学方法分析UCP4蛋白的理化性质和结构特点。利用实时荧光定量PCR技术,对两个具有显著尾型差异的绵羊品种(广灵大尾羊和小尾寒羊)2、4、6、8、10和12月龄共计96个个体的8种组织(大脑、小脑、下丘脑、垂体、皮下脂肪、肾周脂肪、肠系膜脂肪和尾部脂肪)进行mRNA表达研究。【结果】绵羊UCP4基因CDS区长972 bp,编码323个氨基酸,分子量为35.73 kDa,等电点为9.43。二级结构中α螺旋、β折叠股和环分别占56.04%、7.12%和36.84%。该蛋白为跨膜蛋白,无信号肽,但有2个糖基化位点和15个潜在的磷酸化位点。UCP4 mRNA在脑组织和脂肪组织中均有表达,但高表达于脑组织中。品种、月龄和组织对UCP4 mRNA表达均有显著影响。【结论】获得了绵羊UCP4基因的CDS全序列,揭示了其mRNA的表达特征及其影响因素,对进一步研究该基因的结构及其与能量代谢的关系具有重要科学意义。
【Objective】This study aims to clone the coding sequence(CDS) of ovine UCP4 gene,to analyze the CDS and its coding protein structures,and to profile the ontogenetic mRNA expression so that to lay a theoretical foundation for future research on the structure and function of this gene.【Method】The CDS was amplified from ovine cerebrum tissue by reverse transcription-polymerase chain reaction(RT-PCR).Physicochemical features and structural characteristics of ovine UCP4 were analyzed by bioinformatics methods.Ninety-six animals from two sheep breeds with significant differences in tail types(Guangling Large Tailed-and Small Tailed-Han Sheep) were used to study the ontogenetic mRNA expression by real-time quantitative PCR in eight tissues(cerebrum,cerebellum,hypothalamus,pituitary,and subcutaneous fat,perirenal fat,mesenteric fat and tail fat) from 2-to 12-month old age at two-month interval.【Result】The sequencing data showed that the length of complete CDS was 972 bp encoding a protein of 323 amino acids.The isoelectric point(pI) of the protein was 9.43,and the molecular weight was 35.73 kDa.The percentages of helix,strand and loop were 56.04%,7.12% and 36.84%,respectively,in the secondary structure.This transmembrane protein had no signal peptide but with two predicted glycosylation sites and 15 potential phosphorylation sites.The UCP4 mRNA expressed in all these tissues,but overexpressed in brain tissue other than in adipose tissue.Generally,breed,tissue and the months of age had significant influences on the UCP4 mRNA expression.【Conclusion】 The obtained whole CDS of ovine UCP4 gene,expression profile and its limiting factors are of scientific significance to further studies on the relationship between the gene structure and energy metabolism.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第10期2110-2118,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金(30972084)
山西省科技攻关项目(011029)
山西农业大学中青年拔尖创新人才项目(BJRC201203)