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

Abstract

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

CLC Number:

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.

References 21

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Cloning,Bioinformatics Analysis and Construction of Eukaryotic Expression Vector of AIDA Gene in Red Steppe Cattle

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