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棉花学报 ›› 2019, Vol. 31 ›› Issue (2): 121-128.doi: 10.11963/1002-7807.cqcqj.20190307

• 研究与进展 • 上一篇    下一篇

陆地棉HRD转录因子基因原核表达与亚细胞定位分析

陈琴(),曲延英,倪志勇,韩玉慧,程浩然,陈全家*()   

  1. 新疆农业大学农学院/新疆农业大学农业技术重点实验室,乌鲁木齐 830052
  • 收稿日期:2018-07-06 出版日期:2019-03-15 发布日期:2019-03-15
  • 通讯作者: 陈全家 E-mail:cqq0777@163.com;chqjia@126.com
  • 作者简介:陈琴(1982-),女,研究生, cqq0777@163.com
  • 基金资助:
    国家科技重大专项——转基因生物新品种培育(201XZX08005-004)

Prokaryotic Expression and Subcellular Localization Analysis of HRD Transcription Factor Gene in Gossypium hirsutum L.

Chen Qin(),Qu Yanying,Ni Zhiyong,Han Yuhui,Cheng Haoran,Chen Quanjia*()   

  1. College of Agriculture, Xinjiang Agricultural University/Key Laboratory of Agricultural Technology, Xinjiang Agricultural University, Urumqi 830052, China
  • Received:2018-07-06 Online:2019-03-15 Published:2019-03-15
  • Contact: Chen Quanjia E-mail:cqq0777@163.com;chqjia@126.com

摘要:

【目的】AP2/ERF家族基因在植物生长和逆境胁迫中起重要作用,GhHRD转录因子基因是该家族的一员。本试验首次克隆了GhHRD基因,为研究其功能提供理论依据。【方法】选用相对耐旱的新陆中36作为试验材料,通过反转录聚合酶链式反应的方法获得GhHRD基因,并通过生物信息学、原核表达、亚细胞定位和酵母杂交分析预测其结构和功能。【结果】GhHRD基因开放阅读框为555 bp,编码184个氨基酸,相对分子质量为19.539×103 Da,等电点为5,具有典型的AP2/ERF保守结构域。原核表达分析发现,pET-28a(+)-GhHRD可以被诱导表达,且在37 ℃、IPTG浓度1 mmol·L-1条件下高效表达。与GFP融合的重组蛋白GhHRD-GFP定位在细胞核,且具有明显的转录自激活特征。【结论】推测GhHRD可能参与了棉花逆境胁迫应答反应,为进一步探索该转录因子的DNA结合位点和转录调控网络奠定了基础。

关键词: HRD转录因子; 原核表达; 亚细胞定位; 酵母杂交

Abstract:

[Objective] The members of AP2/ERF gene family play an important role in plant growth and stress, and the GhHRD gene is a member of this family. The purpose of our search was to study the function of GhHRD in upland cotton. [Method] The GhHRD gene was cloned by reverse transcription polymerase chain reaction technology and its structure and function were predicted by bioinformatics methods, prokaryotic expression, subcellular localization and yeast hybridization analysis. [Result] The Open read frame of GhHRD gene was 555 bp, encoding 184 amino acids, with relative molecular mass 19.539 kDa, isoelectric point 5, and a typical AP2/ERF conservative domain. The prokaryotic expression analysis showed that expression of pET-28(+)-GhHRD could be efficiently induced under the condition of 37 ℃ and 1 mmol·L-1 IPTG. The recombinant protein GhHRD-GFP is sublocated in the nucleus and has obvious transcriptional self-activation characteristics. [Conclusion] It is speculated that GhHRD may be involved in response to stress responses in cotton. The results laid a foundation for further exploration of DNA binding sites and transcriptional regulatory networks.

Key words: HRD transcriptor factor; prokaryotic expression; subcellular localization; yeast hybridization