猪Nrf2基因克隆、生物信息学分析及启动子区转录活性分析

doi:10.11843/j.issn.0366-6964.2019.07.002

Abstract

Abstract: To understand the structure, function and transcriptional regulation mechanism of Nrf2 gene, six healthy Dapulian pigs, about 90 kg, were selected as experimental animals. The full cDNA sequence and promoter region of Nrf2 gene were cloned by RACE (rapid amplification of cDNA end) and Genome Walking techniques. Biological characteristics of Nrf2 gene and its promoter region were analyzed by bioinformatics softwares. The results showed that the full cDNA sequence of Nrf2 was 2 358 bp, containing 87 bp sequence of 5'UTR, 495 bp sequence of 3'UTR, and 1 776 bp sequence of coding region (CDS) encoded 591 amino acids. The protein molecular weight was 66.402 7 ku, and the isoelectric point (pI) was 4.66. The secondary structure was mainly consisted of α-helix. 2 091 bp DNA sequence of 5' flanking region of Nrf2 gene in Dapulian pig was obtained by the Genome Walking technique. The predicted 5' flanking sequence of the Nrf2 gene contained typical TATA-box, excluding the CpG island. A series of pGL3-Basic expression vectors with different promoter lengths were established. The promoter relative luciferase activities were measured by the dual-luciferase assay system after transient transfection into 293T cells. The results revealed that the region of -903—-710 bp had the highest activity, suggesting that the region of -903—-710 bp existed positive regulatory region or enhancer. Several transcriptional factor binding sites were predicted in this region by bioinformatics methods, which might be involved in Nrf2 gene transcriptional regulation. The complete cDNA sequence and promoter region of Nrf2 gene were successfully obtained. It was found that the region of -903—-710 bp was the core promoter. This study provides a theoretical basis for the genetic improvement of antioxidant stress in pigs.

CLC Number:

S828.2

LIU Zongli, CHEN Tao, YANG Dandan, CUI Jingxiang, LI Chuanhao, ZENG Yongqing, CHEN Wei. Molecular Cloning, Bioinformatics Analysis and Transcriptional Activity of Promoter of Nuclear Factor Erythroid 2-related Factor (Nrf2) Gene in Pig[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(7): 1328-1339.

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Molecular Cloning, Bioinformatics Analysis and Transcriptional Activity of Promoter of Nuclear Factor Erythroid 2-related Factor (Nrf2) Gene in Pig

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