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棉花学报 ›› 2020, Vol. 32 ›› Issue (4): 305-315.doi: 10.11963/1002-7807.yyfpcy.20200703

• 研究与进展 • 上一篇    下一篇

陆地棉苗期低温响应基因GhZAT10的克隆及功能研究

杨永飞1,2,葛常伟2,沈倩2,张思平2,刘绍东2,马慧娟2,陈静2,刘瑞华2,李士丛2,赵新华2,李志坤1,*(),庞朝友2,*()   

  1. 1.河北农业大学/棉花生物学国家重点实验室河北基地,河北 保定 071001
    2.中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南 安阳 455000
  • 收稿日期:2019-12-26 出版日期:2020-07-15 发布日期:2020-07-23
  • 通讯作者: 李志坤,庞朝友 E-mail:lzhk@hebau.edu.cn;chypang@163.com
  • 作者简介:杨永飞(1994―),男,硕士研究生, yfly_yang@126.com
  • 基金资助:
    中国农业科学院科技创新工程(CAAS-ASTIP-2019-ICR)

Cloning and Functional Analysis of Low Temperature Response Gene, GhZAT10, in Upland Cotton at Seedling Stage

Yang Yongfei1,2,Ge Changwei2,Shen Qian2,Zhang Siping2,Liu Shaodong2,Ma Huijuan2,Chen Jing2,Liu Ruihua2,Li Shicong2,Zhao Xinhua2,Li Zhikun1,*(),Pang Chaoyou2,*()   

  1. 1. Hebei Agricultural University/Hebei Base of State Key Laboratory of Cotton Biology, Baoding, Hebei 071001, China
    2. Institute of Cotton Research of the Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China
  • Received:2019-12-26 Online:2020-07-15 Published:2020-07-23
  • Contact: Li Zhikun,Pang Chaoyou E-mail:lzhk@hebau.edu.cn;chypang@163.com

摘要:

【目的】挖掘棉花耐冷相关基因,为培育棉花耐冷品种奠定基础。【方法】克隆陆地棉基因GhZAT10(Zinc finger of Arabidopsis thaliana 10),通过实时荧光定量聚合酶链反应(Quantitative real-time polymerase chain reaction,qRT-PCR)分析冷处理前后该基因在根、茎、叶的表达;通过生物信息学方法分析GhZAT10基因结构及其编码的蛋白质性质及进化关系;通过Gateway技术构建蛋白表达载体35S::GhZAT10-GFP进行亚细胞定位;利用病毒诱导的基因沉默技术研究低温胁迫下GhZAT10在棉花耐冷反应中的功能。【结果】GhZAT10基因开放阅读框长度为813 bp(base pair,碱基对),编码270个氨基酸;GhZAT10在根中表达量高于茎和叶,低温胁迫下在3种组织中均上调表达。GhZAT10蛋白无信号肽,不包含跨膜螺旋,且其活性与其磷酸化调控关系密切。陆地棉GhZAT10与可可、拟南芥、甜橙中的ZAT10蛋白亲缘关系较近;GhZAT10蛋白定位在细胞核;沉默GhZAT10基因使棉花的耐冷能力减弱。【结论】GhZAT10蛋白属于C2H2型锌指蛋白,在陆地棉冷胁迫响应信号途径中具有积极作用。

关键词: 陆地棉; 低温胁迫; GhZAT10; 转录因子

Abstract:

[Objective] The aim of this study is to identify chilling tolerant genes which can provide a basis for breeding upland cotton cultivars with tolerance to chilling. [Method] The GhZAT10 (Zinc finger of Arabidopsis thaliana 10) gene was cloned, and its expression in roots, stems, and leaves was analyzed with cold treatment by quantitative real-time polymerase chain reaction (qRT-PCR). Then we analyzed the structural characteristics of GhZAT10, protein properties and constructed the phylogenetic tree by bioinformatics methods. Next, we constructed subcellular localization vector 35S::GhZAT10-GFP by Gateway technology. Finally, we studied the effect of GhZAT10 in chilling response by virus induced gene silencing of cotton. [Result] The Open reading frame(ORF) length of GhZAT10 gene is 813 bp (base pair), encoding 270 amino acid residues. The expression of GhZAT10 in roots was higher than that in stems and leaves, and up-regulated after chilling stress in these tissues. GhZAT10 does not possess signal peptides or transmembrane helices; its activity is closely related to the phosphorylation regulation. GhZAT10 has homology with the ZAT10 of Theobroma cacao, Arabidopsis thaliana and Citrus sinensis. GhZAT10 protein is located in the nucleus. The GhZAT10-silenced cotton plants were more sensitive to low temperature than wild type. [Conclusion] GhZAT10 belongs to C2H2 zinc finger protein, and plays a positive role to response the chilling stress in upland cotton.

Key words: upland cotton (Gossypium hirsutum L.); low temperature stress; GhZAT10; transcription factor