《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (4): 957-966.doi: 10.16431/j.cnki.1671-7236.2019.04.001

• 生物技术 • 上一篇    下一篇

草原红牛ACSL3基因CDS区克隆、生物信息学分析及组织表达研究

吕阳1,2, 曹阳2, 高一2, 王玉婷1,2, 张国梁2   

  1. 1. 吉林农业大学动物科学技术学院, 长春 130118;
    2. 吉林省农业科学院畜牧科学分院, 公主岭 136100
  • 收稿日期:2018-08-16 出版日期:2019-04-20 发布日期:2019-04-22
  • 通讯作者: 张国梁 E-mail:zgl7777777@163.com
  • 作者简介:吕阳(1991-),女,吉林乾安人,硕士生,研究方向:分子(细胞)遗传与动物育种,E-mail:979286926@qq.com
  • 基金资助:

    国家肉牛牦牛产业技术体系;吉林省农业科技创新工程研究生基金(C7208000418);肉用红牛草原培育;肉牛肉质性状优异基因挖掘与鉴定(20160204006NY);吉林省农业科学院创新工程(CXGC2017JQ001)

Cloning,Bioinformatics and Tissue Expression Analysis of ACSL3 Gene CDS in Red Steppe Cattle

LYU Yang1,2, CAO Yang2, GAO Yi2, WANG Yuting1,2, ZHANG Guoliang2   

  1. 1. College of Animal Sciences and Technology, Jilin Agricultural University, Changchun 130118, China;
    2. Branch of Animal Husbandry, Jilin Academy of Agricultural Science, Gongzhuling 136100, China
  • Received:2018-08-16 Online:2019-04-20 Published:2019-04-22

摘要:

试验旨在克隆草原红牛长链酰基辅酶A合成酶3(long-chain acyl-CoA synthetase 3,ACSL3)基因编码区并对其进行生物信息学分析,同时在mRNA和蛋白质水平上分析ACSL3基因在草原红牛不同组织中的表达差异。利用RT-PCR技术和TA克隆的方法获得草原红牛ACSL3基因CDS序列;利用在线软件对ACSL3基因进行生物信息学分析,分析ACSL3基因与其他物种的同源性并构建系统进化树,分析ACSL3基因编码蛋白质的基本理化性质、潜在磷酸化位点、O-糖基化位点、N-糖基化位点、信号肽、二硫键、跨膜区结构、亚细胞定位及该基因编码蛋白的二级结构、三级结构;通过实时荧光定量PCR和Western blotting技术检测ACSL3基因在草原红牛各组织间的mRNA和蛋白表达水平。结果显示,试验成功克隆了草原红牛ACSL3基因CDS区,全长2 163 bp,编码720个氨基酸,蛋白分子质量为80.28 ku,理论等电点为8.74,属于亲水性蛋白。通过NCBI中BLAST比对发现,草原红牛与牛、绵羊、猪、人、大鼠、小鼠、鸡的ACSL3基因核苷酸序列同源性分别为99%、97%、93%、91%、88%、88%和78%;系统进化树结果表明,草原红牛与牛、绵羊的亲缘关系最近,与鸡的亲缘关系最远。该蛋白序列有7个二硫键,66个磷酸化位点,9个O-糖基化位点,3个N-糖基化位点,不存在信号肽,但存在1个跨膜区。二级结构和三级结构分析结果表明,ACSL3蛋白通过无规则卷曲连接,蛋白质结构以α-螺旋和β-转角为主,为混合型蛋白。mRNA和蛋白表达量检测结果显示,ACSL3基因在肾脏和肌肉组织中表达量较高,显著高于其他组织(P<0.05);在胃、肝脏和心脏中中度表达,显著高于脾脏、肺脏、肠和脂肪(P<0.05);在脾脏、肺脏、肠和脂肪中相对低表达,说明草原红牛ACSL3基因可能与体内脂肪沉积和脂质代谢等调控功能有关。本试验结果为进一步研究ACSL3基因在草原红牛中脂质代谢及脂肪沉积等方面的调控作用提供了基础材料。

关键词: 草原红牛; ACSL3基因; 克隆; 生物信息学; 表达差异; 脂质代谢

Abstract:

This study was aimed to clone the CDS of long-chain acyl-CoA synthetase 3 (ACSL3) gene in Red Steppe cattle,analyze it for bioinformatics,and detect the expression differences in different tissues of Red Steppe cattle at the mRNA and protein levels.The CDS of ACSL3 gene was obtained by RT-PCR and TA cloning methods.A variety of software and online tools were used to analyze the homology among different species and construct phylogenetic tree,analyze physical and chemical properties,potential phosphorylation locus,O-glycosylation sites,N-glycosylation sites,signal peptide,disulfide bonds,transmembrane region,subcellular localization,and the secondary and tertiary structures of ACSL3 protein.Meanwhile,the mRNA and protein expression levels of ACSL3 gene in each tissue of Red Steppe cattle were detected by Real-time PCR and Western blotting.The results showed that the CDS of ACSL3 gene was 2 163 bp in length encoding 720 amino acids with a protein molecular weight of 80.28 ku and a theoretical isoelectric point of 8.74,which was a hydrophilic protein.By NCBI BLAST comparison,the nucleotide sequence homology of ACSL3 gene in Red Steppe cattle shared 99%,97%,93%,91%,88%,88% and 78% identity with Bos taurus,Ovis aries,Sus scrofa,Homo sapiens,Mus musculus,Rattus norvegicus and Gallus gallus,respectively.The phylogenetic tree found that the Red Steppe cattle had the closest relationship with Bos taurus and Ovis aries,and the farthest relationship with Gallus gallus.The protein sequence had 7 disulfide bonds,66 phosphorylation sites,9 O-glycosylation sites,3 N-glycosylation sites,no signal peptide,but had a transmembrane region.The secondary and tertiary structures analysis showed that ACSL3 protein was connected by random coil,and the protein structure was mainly alpha helix and beta turn,which was a mixed protein.Real-time PCR and Western blotting results showed that the ACSL3 mRNA and protein were expressed in all the collected tissues,and the highest expression was found in kidney and muscle tissues,which were significantly higher than that in the other tissues (P<0.05);It was moderately expressed in stomach,liver and heart,which was significantly higher than that in spleen,lung,intestine and fat (P<0.05);While there were the lowest relative expression in spleen,lung,intestine and fat.The results showed that the differential expression levels of ACSL3 gene in Red Steppe cattle might be related to the regulation of fat deposition and lipid metabolism,which provided the basic materials for further study on the regulation of ACSL3 gene on lipid metabolism and fat deposition in Red Steppe cattle.

Key words: Red Steppe cattle; ACSL3 gene; clone; bioinformatics; differential expression; lipid metabolism

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