玉米热激转录因子基因(ZmHsf06)的克隆、表达和定位分析

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摘要热激转录因子(heat shock transcription factors, Hsfs)在植物抵御热胁迫及其他抗逆反应过程中起重要调控作用，之前的研究主要来自模式植物拟南芥(Arabidopsis thaliana)和番茄(Lycopersicon esculentum)，在玉米(Zea mays)中研究尚少。本研究通过同源基因克隆的方法，从42 ℃热胁迫1 h的玉米幼叶中克隆了玉米热激转录因子基因ZmHsf06(GenBank登录号: GRMZM2G115456_T01)，并对其在玉米不同器官中的表达水平及亚细胞定位进行了分析。结果显示，ZmHsf06编码序列全长1 584 bp，编码527个氨基酸,包含典型的Hsf家族DNA结合结构域。序列分析表明，该蛋白含有核定位信号序列KKRR，核输出信号序列IGDLTEQM和序列为DSFWEQFL的AHA(aromatic, large hydrophobicand acidic amino residues)激活结构域。正常生长条件下，ZmHsf06在玉米幼苗的根系、茎和叶片以及开花期的功能叶、幼穗、幼胚和花粉中均有不同程度的表达。幼苗期根中的表达量相对较高；开花期在花粉中表达最高，功能叶中最低。42 ℃热胁迫、外源脱落酸(abscisic acid, ABA)和盐胁迫处理均能上调ZmHsf06的表达。通过在洋葱(Allium cepa L.)内表皮瞬时表达融合蛋白发现，正常条件下ZmHsf06定位在细胞核，37 ℃热激后也只在细胞核中观察到GFP荧光。推测玉米ZmHsf06可能在转录水平上介导玉米花粉发育，参与了对多种逆境胁迫的调控过程，并且在细胞核内行使上述功能。本研究为全面克隆和了解玉米热激转录因子家族基因，并进一步阐述其生物学功能提供理论依据。

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	李慧聪
	李国良
	郭秀林

关键词 ：玉米, ZmHsf06, 热激转录因子, 逆境胁迫, 花粉发育

Abstract：Heat shock transcription factors (Hsfs) widely exist in plants and play a key role under extreme environmental conditions, especially under heat shock stress. Most researches focused on model plants such as Arabidopsis and tomato (Lycopersicon esculentum). There were few reports about maize Hsfs so far. In this study, a Hsf gene, named ZmHsf06 (GenBank accession: GRMZM2G115456_T01), was cloned from maize (Zea mays) young leaves treated by heat shock at 42 ℃ for 1 h using homologous cloning methods. The patterns of ZmHsf06 expression level in different organs and its subcellular location were analyzed. Sequence analysis showed that the coding sequences (CDS) of ZmHsf06 was 1 584 bp encoding a protein of 527 amino acids. ZmHsf06 contained not only the most conserved and typical DNA-binding domain (DBD) of Hsf family, but also other functional domains such as a nuclear localization signal (NLS) KKRR peptide, a nuclear export signal (NES) IGDLTEQM peptide and an aromatic, large hydrophobic and acidic amino residues (AHA) DSFWEQFL peptide. Real-time quantitative PCR (qRT-PCR) analysis showed that under normal growth conditions, ZmHsf06 expressed in roots, stems and leaves of maize seedlings with the highest expression level in roots, and also in functional leaves, ears, immature embryo and pollen at anther period with the highest expression level in pollen and the lowest in leaves. The expression level of ZmHsf06 was up-regulated by 42 ℃ heat shock, abscisic acid(ABA) and salt stress, respectively. In details, under heat shock of 42 ℃, the highest expression level of ZmHsf06 in roots was 16 folds of that in leaves, and it appeared later than in leaves. While treated with ABA, the highest expression level in leaves was 2 folds of that in roots, and it appeared later than in roots. Under salt stress, the expression levels of ZmHsf06 in leaves and roots were increased significantly and shared the similar patterns. Based on transient expression assay using onion (Allium cepa L.) epidermis, it was found that the ZmHsf06 was located in nucleus specifically both under normal condition and heat shock at 37 ℃ within 1 h. It suggested that ZmHsf06 probably participates in signal transduction process of pollen development and responses to abiotic stresses at transcription level, and functions in nucleus. The results of this work provide basic data for isolating and characterizing more Hsfs in maize, as well as exploring their biological functions in abiotic stresses.

Key words： Maize ZmHsf06 Heat shock transcription factor Abiotic stress Pollen development

收稿日期: 2014-07-21 出版日期: 2015-01-06

基金资助:河北省应用基础研究重点研究计划项目

通讯作者: 郭秀林 E-mail: myhf2002@163.com

引用本文:

李慧聪李国良郭秀林. 玉米热激转录因子基因(ZmHsf06)的克隆、表达和定位分析[J]. , 2015, 23(1): 41-51.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I1/41

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