草原红牛AIDA基因克隆、生物信息学分析及真核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2019.08.001

摘要/Abstract

摘要：

本研究旨在对草原红牛AIDA基因进行克隆、生物信息学分析和差异表达研究，并构建真核表达载体，以期在细胞水平上探究AIDA基因对牛前体脂肪细胞分化的影响。应用RT-PCR方法从草原红牛脂肪组织中扩增AIDA基因编码区，测序鉴定后对其核苷酸和氨基酸序列进行生物信息学分析，同时利用实时荧光定量PCR技术研究AIDA基因在草原红牛9个组织（心脏、肝脏、脾脏、肺脏、肾脏、胃、肠、肌肉、脂肪）和前体脂肪细胞成脂分化过程中的表达规律；构建真核表达载体pBI-CMV3-AIDA，转染草原红牛前体脂肪细胞，通过实时荧光定量PCR方法检测AIDA基因在mRNA水平上的表达情况。结果显示，AIDA基因编码区全长921 bp，编码306个氨基酸，含有4个潜在的糖基化位点和29个潜在的磷酸化位点；亚细胞定位主要分布于细胞质、细胞核和线粒体上。AIDA基因在草原红牛9个组织中均有表达，其中在肾脏组织中表达量最高，显著高于其他组织（P<0.05）。成脂分化结果表明，AIDA基因mRNA表达量在分化的第2天达到最高，随着脂肪细胞的成熟，其表达量逐渐降低；双酶切及测序结果表明，试验成功构建了AIDA基因的真核表达载体pBI-CMV3-AIDA，且过表达组AIDA基因mRNA表达量极显著高于对照组（P<0.01）。本试验成功构建了AIDA基因真核表达载体，并在草原红牛前体脂肪细胞中高度表达，该结果为体外研究牛AIDA基因对脂肪合成代谢及其机体代谢的调节机制提供了基础材料。

关键词: AIDA基因; 草原红牛; 前体脂肪细胞; 真核表达载体

Abstract:

This study was aimed to clone, bioinformatics and differential expression analyze of AIDA gene in Red Steppe cattle, and construct a eukaryotic expression vector to explore the effects of AIDA genes on the differentiation of bovine preadipocytes at the cellular level.The AIDA gene coding region was amplified from the adipose tissue of Red Steppe cattle by RT-PCR.The nucleotide and amino acid sequences were analyzed by bioinformatics after sequencing.The mRNA expression levels of AIDA gene in 9 tissue of Red Steppe cattle (heart, liver, spleen, lung, kidney, stomach, intestine, muscle and fat) and expression patterns of adipogenic differentiation of preadipocytes were detected by Real-time PCR;The eukaryotic expression vector pBI-CMV3-AIDA was constructed, and transfected into preadipocytes of Red Steppe cattle.The expression of AIDA gene mRNA was detected by Real-time PCR.The results showed that the sequence of AIDA gene CDS was 921 bp, encoding 306 amino acids, containing 4 potential glycosylation sites and 29 potential phosphorylation sites.The subcellular localization was mainly distributed in the cytoplasm, nucleus and mitochondria.AIDA gene was expressed in all 9 tissues of Red Steppe cattle, and the expression was the highest in kidney, which was significantly higher than that in other tissues (P<0.05).The adipogenic differentiation results showed that the expression of AIDA gene mRNA reached the highest on the second day of differentiation, and the expression of AIDA gene gradually decreased with the maturation of adipocytes.The results of double digestion and sequencing showed that the eukaryotic expression vector pBI-CMV3-AIDA of AIDA gene was successfully constructed, and the expression of AIDA gene mRNA in overexpression group was extremely significantly higher than control group (P<0.01).In this experiment, AIDA eukaryotic expression vector was successfully constructed and highly expressed in bovine preadipocytes, which provided a basis for in vitro study of the regulation mechanism of bovine AIDA gene on fat anabolism and metabolism.

Key words: AIDA gene; Red Stepppe cattle; preadipocytes; eukaryotic expression vector

中图分类号:

吕阳, 曹阳, 高一, 刘理想, 薛佳佳, 张国梁. 草原红牛AIDA基因克隆、生物信息学分析及真核表达载体的构建[J]. 《中国畜牧兽医》, 2019, 46(8): 2193-2202.

LYU Yang, CAO Yang, GAO Yi, LIU Lixiang, XUE Jiajia, ZHANG Guoliang. Cloning,Bioinformatics Analysis and Construction of Eukaryotic Expression Vector of AIDA Gene in Red Steppe Cattle[J]. , 2019, 46(8): 2193-2202.

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