陆地棉苗期低温响应基因GhZAT10的克隆及功能研究  

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

作　　者：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[1] 庞朝友[2] Yang Yongfei;Ge Changwei;Shen Qian;Zhang Siping;Liu Shaodong;Ma Huijuan;Chen Jing;Liu Ruihua;Li Shicong;Zhao Xinhua;Li Zhikun;Pang Chaoyou(Hebei Agricultural University/Hebei Base of State Key Laboratory of Cotton Biology,Baoding,Hebei 071001,China;Institute of Cotton Research of the Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology,Anyang,Henan 455000,China)

机构地区：[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

出　　处：《棉花学报》2020年第4期305-315,共11页Cotton Science

基　　金：中国农业科学院科技创新工程(CAAS-ASTIP-2019-ICR)。

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Gh ZAT10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。[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.

关 键 词：陆地棉 低温胁迫 GhZAT10 转录因子 

分 类 号：S562[农业科学—作物学]