猪Nrf2基因克隆、生物信息学分析及启动子区转录活性分析  

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

作　　者：刘宗立 陈涛[1,2] 杨丹丹 崔景香 李川皓[1,2] 曾勇庆 陈伟[1,2] LIU Zongli;CHEN Tao;YANG Dandan;CUI Jingxiang;LI Chuanhao;ZENG Yongqing;CHEN Wei(College of Animal Science and Technology,Shandong Agricultural University,Tai’an 271018,China;Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention,Tai’an 271018,China;Weifang University of Science and Technology,Weifang 262700,China)

机构地区：[1]山东农业大学动物科技学院,泰安271018 [2]山东省动物生物工程与疾病防治重点实验室,泰安271018 [3]潍坊科技学院,潍坊262700

出　　处：《畜牧兽医学报》2019年第7期1328-1339,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31401055);山东省现代农业产业技术体系生猪创新团队建设专项(SDAIT-08-02);山东省“双一流”奖补资金项目(SYL2017YSTD12);山东省农业良种工程重大课题(2013LZ01-003);山东农业大学青年科技创新基金

摘　　要：旨在了解猪Nrf2基因的结构和功能,研究Nrf2基因的转录调控机制。本研究选取6头90 kg左右的健康大蒲莲猪为试验动物,采用cDNA末端快速克隆技术(RACE)和染色体步移技术获得大蒲莲猪Nrf2基因完整的cDNA序列和启动子区域,利用生物信息学软件分析Nrf2基因及其启动子区域的生物学特征。结果,Nrf2基因cDNA全长2 358 bp,包括87 bp的5′UTR,495 bp的3′UTR序列,CDS区大小为1 776 bp,编码591个氨基酸。对预测蛋白质序列进行生物信息学分析,蛋白分子量为66.402 7 ku,理论等电点(pI)为4.66,大部分二级结构为α-螺旋。采用染色体步移技术扩增得到5′侧翼序列2 091 bp的片段。Nrf2基因5′侧翼区经预测含有典型的TATA-box,不含CpG岛。构建不同长度启动子片段的pGL3-Basic表达载体,转染293T细胞后经双荧光素酶活性检测,提示在-903^-710 bp区域内可能存在正调控区或增强子,生物信息学预测其中含有多个转录因子结合位点,可能参与Nrf2基因转录调控。本研究成功获得了Nrf2基因完整的cDNA序列和启动子区域,并且发现-903^-710 bp为核心启动子,本研究为猪抗氧化应激的遗传改良提供一定理论基础。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 293 T 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.

关 键 词：猪 Nrf2基因 基因克隆 启动子 双荧光素酶 

分 类 号：S828.2[农业科学—畜牧学]