陆地棉HRD转录因子基因原核表达与亚细胞定位分析

doi:10.11963/1002-7807.cqcqj.20190307

摘要/Abstract

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

陈琴,曲延英,倪志勇,韩玉慧,程浩然,陈全家. 陆地棉HRD转录因子基因原核表达与亚细胞定位分析[J]. 棉花学报, 2019, 31(2): 121-128.

Chen Qin,Qu Yanying,Ni Zhiyong,Han Yuhui,Cheng Haoran,Chen Quanjia. Prokaryotic Expression and Subcellular Localization Analysis of HRD Transcription Factor Gene in Gossypium hirsutum L.[J]. Cotton Science, 2019, 31(2): 121-128.

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链接本文: http://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/1002-7807.cqcqj.20190307

http://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2019/V31/I2/121

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