山羊Hoxa5基因克隆及表达特性  

Cloning and Expression Characteristics of Hoxa5 Gene in Goat

作　　者：陈定双 王瑞龙 林亚秋[1,2] 王永[1,2] 朱江江[1,2] 李鑫[1,2] 张浩 李艳艳 CHEN Dingshuang;WANG Ruilong;LIN Yaqiu;WANG Yong;ZHU Jiangjiang;LI Xin;ZHANG Hao;LI Yanyan(Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Ministry of Education,Sichuan Province,Southwest Minzu University,Chengdu 610041,China;College of Animal & Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部四川省重点实验室,四川成都610041 [2]西南民族大学畜牧兽医学院,四川成都610041

出　　处：《华北农学报》2021年第6期219-227,共9页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(82002817);西南民族大学研究生创新型科研项目(CX2021SZ69)。

摘　　要：Hoxa5是HOX家族中的一员,在生长发育与肿瘤发生发展过程中发挥着重要作用,但其在反刍动物中的研究较少。为了获得山羊Hoxa5基因序列,明确其表达特征,知悉其在山羊不同组织的表达水平,利用RT-PCR技术,从简州大耳羊的皮下脂肪组织中克隆得到Hoxa5基因序列,用生物信息学的方法分析其生物学特性,用实时荧光定量PCR技术检测Hoxa5基因在简州大耳羊心、肝、脾、肺、肾、背、股、臂等10个组织中的表达情况。结果表明:山羊Hoxa5基因的开放阅读框为813 bp,共编码270个氨基酸。Hoxa5相对分子质量为29.283 ku,等电点为9.42,无信号肽和跨膜结构域,该蛋白属于非分泌蛋白;Hoxa5蛋白的二级结构预测发现,Hoxa5蛋白中无规卷曲含量最高,其次是α螺旋,β转角占总氨基酸数相对较少;氨基酸同源性比对结果显示,简州大耳羊与绵羊、牛、马、猪、黑鼠、豚鼠、小鼠及人的Hoxa5氨基酸序列相似性依次为100.00%,99.63%,99.26%,99.26%,98.89%,98.89%,98.89%和98.15%,说明该基因在不同的物种间具有较高的保守性;实时荧光定量PCR结果显示,Hoxa5基因在山羊的组织中差异表达,在肾脏中表达量最高;构建pEGFP-Hoxa5融合载体转染山羊皮下脂肪细胞后的荧光定位显示该基因主要定位于细胞核中。研究表明,山羊Hoxa5基因在皮下脂肪细胞分化过程上调且蛋白定位于细胞核中,推测该基因可能作为转录因子调控山羊皮下脂肪细胞分化。Hoxa5 is a member of the HOX family and plays an important role in the process of growth,development and tumorigenesis.However,there is less research on it in ruminants.In order to obtain the goat Hoxa5 gene sequence,to clarify its expression characteristics and know its expression level in different tissues of goats.The Hoxa5 gene was cloned from the subcutaneous fat tissue of Jianzhou big ear sheep using RT-PCR technology,and its biological characteristics were analyzed by bioinformatics methods.Real-time fluorescent quantitative PCR technology was used to detect the expression of Hoxa5 gene in the heart,liver,spleen,lung,kidney,back,thigh,arm,etc.tissues of Jianzhou big ear sheep.The results showed that the open reading frame of the goat Hoxa5 gene was 813 bp,encoding a total of 270 amino acids.the relative molecular mass of Hoxa5 was 29.283 ku,the isoelectric point was 9.42,and there was no signal peptide and transmembrane domain.This protein was a non-secreted protein;the secondary structure prediction of Hoxa5 protein found that the content of random coils in Hoxa5 protein was the highest,followed byαhelix,andβturn accounts for a relatively small number of amino acids;amino acid homology comparison results showed that the Hoxa5 amino acid sequence similarity between Jianzhou big ear sheep and Ovis aries,Bos taurus,Equus caballus,Sus scrofa,Rattus rattus,Cavia porcellus,Mus musculus and Homo sapiens were 100.00%,99.63%,99.26%,99.26%,98.89%,98.89%,98.89%and 98.15%.The results of Real-time fluorescent quantitative PCR showed that Hoxa5 gene was differentially expressed in selected tissues,with the highest expression level in the kidney;Fluorescence localization of pEGFP-Hoxa5 fusion vector transfected into goat subcutaneous adipocyte showed that the gene was mainly located in the nucleus.This study showed that the goat Hoxa5 gene was up-regulated during subcutaneous adipocyte differentiation and the protein was localized in the nucleus.The results suggested that this gene might act as a transcription factor

关 键 词：山羊 Hoxa5 表达特征 核定位 

分 类 号：Q78[生物学—分子生物学] S826[农业科学—畜牧学]