《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (8): 1975-1982.doi: 10.16431/j.cnki.1671-7236.2016.08.007

• 生物技术 • 上一篇    下一篇

水牛Keap1基因的克隆、序列分析及组织表达研究

赵鑫, 阮子芸, 郭振伟, 周文婷, 秦希玲, 牛向丽, 陆凤花, 石德顺   

  1. 广西大学, 亚热带农业生物资源保护与利用国家重点实验室, 南宁 530004
  • 收稿日期:2016-01-18 出版日期:2016-08-20 发布日期:2016-08-23
  • 通讯作者: 陆凤花, 石德顺 E-mail:lfhgggg@163.com;ardsshi@gxu.edu.cn
  • 作者简介:赵鑫(1989-),男,陕西宝鸡人,硕士生,研究方向:动物繁殖新技术与动物胚胎工程,E-mail:272277845@qq.com
  • 基金资助:

    国家高技术研究发展计划“863”项目(2011AA100607);国家自然科学基金项目(31560633);广西科学基金(2012GXNSFFA060004);广西研究生教育创新计划项目(YCBZ2015004);广西计生委科研项目(桂人口计生研1109)

Cloning and Sequence Analysis of Buffalo Keap1 Gene and Investigation of Its Expression Pattern in Different Tissues

ZHAO Xin, RUAN Zi-yun, GUO Zhen-wei, ZHOU Wen-ting, QIN Xi-ling, NIU Xiang-li, LU Feng-hua, SHI De-shun   

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China
  • Received:2016-01-18 Online:2016-08-20 Published:2016-08-23

摘要:

本研究克隆了水牛Keap1基因的全长编码区,并对其序列进行了生物信息学分析,同时探索了其在水牛各组织中的表达差异。根据GenBank中公布的牛Keap1基因的序列信息,设计特异性引物并扩增出水牛Keap1的目的片段,利用生物信息学分析方法,对测序所得水牛Keap1基因序列、预测蛋白质序列进行了分析,并采用实时荧光定量PCR技术对Keap1基因mRNA在水牛各组织中的表达进行了研究。结果表明,水牛Keap1基因编码区全长1 875 bp,预测编码624个氨基酸;多重分析结果显示,水牛与牛、绵羊、野猪和人Keap1基因的同源性分别为99%、96%、92%和90%;进化树分析表明Keap1在物种间具有较高保守性,不同物种间Keap1序列的差异符合物种间的进化性。对Keap1蛋白质二级结构预测发现,其包含24个α-螺旋、40个β-螺旋、38个T转角和27个无规则卷曲。实时荧光定量PCR结果显示,Keap1基因在水牛的心脏、肝脏、脾脏、肺脏、肾脏、卵巢和肌肉组织中均有表达,但心脏中表达量最高,肝脏、脾脏中表达量较低。本研究成功克隆了水牛Keap1基因,并进行了相关生物信息学分析及其mRNA在水牛各组织的表达情况研究,为阐明Keap1-Nrf2-ARE信号通路,提高水牛胚胎体外培养的抗氧化能力奠定基础。

关键词: 水牛; Keap1基因; 克隆; 分析; 表达

Abstract:

In this study,the CDS sequence of buffalo Keap1 gene was cloned and analyzed,then its expression pattern in different tissues was also investigated.A pair of primers of buffalo Keap1 gene was designed based on the nucleotide sequence of Bos taurus Keap1 gene from GenBank,and then the buffalo Keap1 gene was amplified.Using the bioinformation techniques,the gene sequence and the protein structure were analyzed.The expression level of Keap1 gene in different tissues were detected with Real-time quantitative PCR.The results showed that the length of buffalo Keap1 gene coding sequence was 1 875 bp and encoded 624 amino acids.The multiple sequence alignment results showed that buffalo Keap1 gene shared 99%,96%,92% and 90% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa and Homo sapiens,respectively.And the phyogenetic tree also showed the conservatism between several different species.The second structure of buffalo Keap1 protein was predicted as 24 alpha regions,40 beta regions,38 turn regions and 27 coil regions.In addition,the results of Real-time quantitative PCR showed that Keap1 mRNA exists in all seven tissues,but the most abundant expression was in heart and the minimal expression was in liver and spleen.The results provided an foundation for further study of Keap1-Nrf2-ARE signal pathway,for enhancing the ability of antioxidant of buffalo embryo in vitro culture.

Key words: buffalo; Keap1 gene; clone; analysis; expression

中图分类号: