猪Nrf2基因克隆、表达及序列特征分析

Cloning,Expression and Sequence Characterization Analysis of Nrf2 Gene of Pig(Sus scrofa domesticus)

Nrf2是细胞抗氧化体系Keap1-Nrf2/ARE中的关键转录因子。本研究以杜长大仔猪肝脏总RNA为材料,根据GenBank公布的人、牛和大鼠Nrf2序列设计引物,利用RT-PCR法克隆猪Nrf2基因的全编码序列,并通过生物信息学分析预测该基因的结构和系统发育;采用qRT-PCR技术检测Nrf2基因在仔猪不同生理阶段和不同组织中的表达情况。结果表明:猪Nrf2基因包含1个1776 bp的开放阅读框,编码591个氨基酸。生物信息学分析显示,猪Nrf2蛋白含3个特征结构域,与牛Nrf2的氨基酸序列相似性最高,为88.45%,而与褶纹冠蚌相似度最低,为30.64%。qRT-PCR结果显示,Nrf2在本实验所检测的组织中均有表达,其中以背最长肌表达量最高,显著高于其他组织(p<0.05);心脏和肾脏次之,小肠和肝脏表达较少;肺脏和脾脏表达丰度最低;Nrf2在初生、断奶、35日龄、70日龄和100日龄猪的背最长肌中均有表达,以断奶阶段表达量最高(p<0.05),100日龄表达量最低(p<0.05)。本研究为进一步探讨猪Nrf2基因的生物学功能提供了理论依据。

Nrf2 is a key transcription factor in the cellular antioxidant system Keap1-Nrf2/ARE.In this study,the total RNA from the liver of piglets was used as the material,and primers were designed according to the Homo sapiens,Bos taurus and Rattus norvegicus Nrf2 sequences published by GenBank.The full coding sequence of pig Nrf2 gene was cloned by RT-PCR,and the structure and phylogeny of the gene were predicted by bioinformatics analysis.The expression of Nrf2 gene in different physiological stages and tissues of piglets was detected by qRT-PCR.The results showed that the sequence of cDNA of pig Nrf2 containing a 1776 bp of open reading frame which encoded 591 amino acids.Structure analysis of amino acid showed that Nrf2 contains three characteristic domains,and the amino acid sequence similarity between pig Nrf2 and Bos taurus Nrf2 was the highest(88.45%),while the similarity between pig Nrf2 and Cristariaplicata was the lowest(30.64%).The qRT-PCR revealed that the pig Nrf2 expressed in all 7 tissues examined.The Nrf2 mRNA expression level in longissimus muscle was highest,which was significantly higher than that in other tissues(p<0.05),followed by heart and kidney,and relative lower expression was detected in liver and small intestine,and the lowest expression was found in lung and spleen.