陆地棉苗期低温响应基因GhZAT10的克隆及功能研究

doi:10.11963/1002-7807.yyfpcy.20200703

摘要/Abstract

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

杨永飞,葛常伟,沈倩,张思平,刘绍东,马慧娟,陈静,刘瑞华,李士丛,赵新华,李志坤,庞朝友. 陆地棉苗期低温响应基因GhZAT10的克隆及功能研究[J]. 棉花学报, 2020, 32(4): 305-315.

Yang Yongfei,Ge Changwei,Shen Qian,Zhang Siping,Liu Shaodong,Ma Huijuan,Chen Jing,Liu Ruihua,Li Shicong,Zhao Xinhua,Li Zhikun,Pang Chaoyou. Cloning and Functional Analysis of Low Temperature Response Gene, GhZAT10, in Upland Cotton at Seedling Stage[J]. Cotton Science, 2020, 32(4): 305-315.

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链接本文: https://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/1002-7807.yyfpcy.20200703

https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2020/V32/I4/305

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引物名称 Primer name	正向引物（5'-3'） Forward primer (5'-3')	反向引物（5'-3'） Reverse primer (5'-3')
pGhZAT10	ATGGCTTTAGAAGCTCTCAACTC	ATTTGTTTCGTTGACTTGCATTT
pGVZAT10	AGCGCCAGCGGCGTGACGACA	GTGGCATCCGCCCCAAGAGAC
pGqZAT10	TCACCAAGCGTAAGCGTTCCAA	GAACGATGGCGGTGTGGGATAA
pGhActin7	TATGTTGCCATCCAGGCCGTTC	AACACCATCACCGGAATCCAGC