秦川肉牛AdipoR1和AdipoR2基因克隆、生物信息学分析及表达研究

doi:10.16431/j.cnki.1671-7236.2021.01.003

摘要/Abstract

摘要： 试验旨在利用生物信息学方法对秦川牛肉用新品系（以下简称"秦川肉牛"）AdipoR1、AdipoR2基因的CDS区进行克隆及分析，并探究其在秦川肉牛不同组织及肌细胞诱导分化不同时间的表达情况。以秦川肉牛为研究对象，经PCR扩增得到AdipoR1、AdipoR2基因CDS区序列，运用生物信息学软件对其功能结构进行预测，同时采用实时荧光定量PCR术获得AdipoR1、AdipoR2基因在秦川肉牛15个组织和诱导分化后不同时间的表达量，再进行差异分析。结果显示，秦川肉牛AdipoR1基因编码序列全长为1 128 bp，编码375个氨基酸，蛋白分子质量为42 446.41 u，理论等电点为6.70，AdipoR1蛋白二级结构主要由α-螺旋构成，二、三级结构预测结果一致，属于亲水性蛋白，没有信号肽位点；AdipoR2基因编码序列全长1 161 bp，编码386个氨基酸，蛋白分子质量为43 707.70 u，理论等电点为6.27。亚细胞定位结果表明，AdipoR1蛋白有60.9%的可能定位在细胞膜，AdipoR2蛋白有73.9%的可能定位在细胞膜。不同物种氨基酸系统进化树结果显示，秦川肉牛AdipoR1、AdipoR2基因编码的氨基酸序列分别与瘤牛和野牦牛的亲缘性最近。实时荧光定量PCR结果显示，AdipoR1、AdipoR2基因在秦川肉牛心脏、肝脏、肌肉等15个组织中均有表达，且在肌肉组织中的表达量最高，极显著高于其他组织（P<0.01），在肌细胞诱导分化时序上也有明显差异。本试验揭示了AdipoR1和AdipoR2基因在秦川肉牛不同组织和肌细胞诱导分化后不同时间上的表达差异，以期为进一步探究AdipoR1、AdipoR2基因的生物学功能与调控机理奠定基础。

关键词: 秦川肉牛; AdipoR1基因; AdipoR2基因; 基因克隆; 组织表达

Abstract: The purpose of the experiment was to use bioinformatics to clone and analyze of the CDS regions of the AdipoR1 and AdipoR2 genes of the new Qinchuan beef line (hereinafter referred to as "Qinchuan beef cattle"),and to explore its expression in different tissues and muscle cells of Qinchuan beef cattle induced differentiation at different time.Taking Qinchuan beef cattle as the research object,the CDS region sequences of AdipoR1 and AdipoR2 genes were amplified by PCR,and their functional structure was predicted using bioinformatics software.At the same time,the expression level of AdipoR1 and AdipoR2 genes in 15 tissues of beef cattle and different time after induction differentiation was obtained using qRT-PCR and analyzed for differences then.The results showed that the full length of AdipoR1 gene coding sequence of Qinchuan beef cattle was 1 128 bp,encoding 375 amino acids,the protein molecular weight was 42 446.41 u,and the theoretical isoelectric point was 6.70.The secondary structure of AdipoR1 protein was mainly composed of α-helix.The prediction results of the secondary and tertiary structures were consistent.AdipoR1 protein was hydrophilic protein and had no signal peptide site.The full length of the AdipoR2 gene coding sequence was 1 161 bp,encoding 386 amino acids,the protein molecular weight was 43 707.70 u,and the theoretical isoelectric point was 6.27.The subcellular localization results showed that AdipoR1 protein was 60.9% likely to be located on the cell membrane,and AdipoR2 protein was 73.9% likely to be located on the cell membrane.The results of the amino acids phylogenetic tree of different species showed that the amino acid sequences encoded by the AdipoR1 and AdipoR2 genes of Qinchuan beef cattle were closest to Bos indicus and Bos mutus,respectively.qRT-PCR results showed that AdipoR1 and AdipoR2 genes were all expressed in 15 tissues of Qinchuan beef cattle such as heart,liver,muscle,and so on.And the expression level in muscle tissues was the highest,which was significantly higher than other tissues (P<0.01).There were also obvious differences in the timing of muscle cell differentiation.The experiment revealed the differences in the expression of AdipoR1 and AdipoR2 genes in different tissues and muscle cells of Qinchuan beef cattle at different time after induction differentiation,in order to lay a foundation for further exploration of the biological functions and regulatory mechanisms of AdipoR1 and AdipoR2 genes.

Key words: Qinchuan beef cattle; AdipoR1 gene; AdipoR2 gene; gene cloning; tissue expression

中图分类号:

S823.8⁺1

陈星伊, 杨昕冉, 孙冰, 昝林森. 秦川肉牛AdipoR1和AdipoR2基因克隆、生物信息学分析及表达研究[J]. 中国畜牧兽医, 2021, 48(1): 22-34.

CHEN Xingyi, YANG Xinran, SUN Bing, ZAN Linsen. Cloning,Bioinformatics Analysis and Expression of AdipoR1 and AdipoR2 Genes from Qinchuan Beef Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 22-34.

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