烟草NtWRKY71基因克隆与表达分析

Cloning and expression analysis of NtWRKY71 of Nicotiana tabacum

【背景和目的】WRKY转录因子作为一类植物特有蛋白,不仅参与植物抗病、抗逆等防御反应,而且在植物分枝发育过程中发挥重要作用。为鉴定烟草分枝发育相关的WRKY转录因子。【方法】以拟南芥分枝发育相关转录因子AtWRKY71序列为参考,在烟草基因组中进行鉴定和克隆。利用生物信息学方法分析烟草NtWRKY71理化性质和进化关系。通过qPCR和芯片表达谱分析了NtWRKY71时空表达特征和激素诱导表达模式,利用本氏烟草瞬时表达系统进行亚细胞定位分析。【结果】烟草NtWRKY71基因的蛋白编码序列为930bp,编码一个由309个氨基酸组成的WRKY蛋白。烟草NtWRKY71含有保守的WRKY蛋白结构域。进化分析显示NtWRKY71与野生烟草WRKY71、土豆WRKY71以及拟南芥WRKY71进化关系较近。基因表达分析表明,NtWRKY71在雄蕊、雌蕊以及根中大量表达;另外,NtWRKY71受赤霉素、生长素和水杨酸的诱导上调表达。亚细胞定位实验表明,NtWRKY71定位在细胞核中。【结论】烟草NtWRKY71为拟南芥分枝调控基因AtWRKY71的同源基因,其表达受到多种激素的调控。研究结果为深入解析NtWRKY71在烟草分枝发育中的功能奠定了基础。

As a class of plant-specific proteins,WRKY transcription factors not only participate in plant defense reactions such as disease resistance and stress resistance,but also play an important role in plant branching development.To identify the WRKY transcription factor associated with tobacco branching development,the Arabidopsis AtWRKY71 was cloned and used as the reference sequence to identify its homologous sequence in the tobacco genome.The physicochemical properties and evolutionary relationships of NtWRKY71 in tobacco were analyzed by bioinformatics methods.The spatiotemporal expression features,hormone-induced expression patterns were analyzed by qPCR and microarray expression profile data.Subcellular localization analysis was performed using the tobacco transient expression system.The protein coding sequence of tobacco NtWRKY71 is 930 base pairs,which encodes a WRKY protein consisting of 309 amino acids.Tobacco NtWRKY71 contains a conserved WRKY domain.Evolutionary analysis showed that NtWRKY71 was closely related to wild tobacco WRKY71,potato WRKY71 and Arabidopsis WRKY71.Gene expression analysis showed that NtWRKY71 was highly expressed in stamens,pistils and roots.In addition,the expression of NtWRKY71 was up-regulated by GA,auxin and salicylic acid.Subcellular localization experiments showed that NtWRKY71 was localized in the nucleus.Tobacco NtWRKY71 is a homolog of the Arabidopsis branching regulatory gene AtWRKY71,whose expression is regulated by multiple hormones.These results provide a theoretical basis for a thorough analysis of the function of NtWRKY71 in tobacco branching development.