玉米逆境胁迫响应基因ZmbZIP84的克隆与功能验证

Cloning and Functional Verification of Maize Stress Response Gene ZmbZIP84

碱性亮氨酸拉链(Basic leucine zipper,bZIP)转录因子是真核生物中分布最广泛、结构极其保守的家族之一,在植物生长进程中发挥重要作用。为验证bZIP转录因子在逆境胁迫中发挥的作用,本研究通过分析转录组数据,筛选到27个参与干旱-复水胁迫的bZIP基因,计算相关性系数、构建共表达网络图发现ZmbZIP84(Zm00001d053988)是核心节点基因;该基因位于玉米第4号染色体,具有高度保守的bZIP结构域;分析理化性质,发现该基因是亲水性蛋白且属于不稳定蛋白;构建系统发育树,发现该基因与柳枝稷和高粱的亲缘关系最近;实时荧光定量PCR(qRT-PCR)结果显示,ZmbZIP84在玉米各组织中均有表达,成熟根中的表达量最高;设置20%聚乙二醇(PEG)-6000、42℃高温、200 mmol·L^(-1)NaCl以及缺乏铵态氮等模拟试验,发现该基因在高温、干旱、氮处理下表达量显著上调,而在NaCl处理下表达量下调,说明ZmbZIP84基因积极参与并响应非生物胁迫;利用CRISPR/Cas9技术获得ZmbZIP84基因的拟南芥同源基因缺失型纯合突变体,发现在高温、干旱胁迫处理下突变体植株生长受到严重抑制、叶片萎蔫、甚至干死,而野生型植株影响较小,说明在干旱、高温胁迫处理下,ZmbZIP84基因敲除后抗旱、耐高温能力下降;亚细胞定位发现该基因编码的蛋白位于细胞核。本研究结果可为后续研究ZmbZIP84基因的功能及下游靶基因的调控机制奠定基础,为培育抗逆性玉米新品种提供参考。

Basic leucine zipper(bZIP)transcription factors are one of the most widely distributed and structurally conserved families in eukaryotes and play important roles in plant growth processes.To verify the role of bZIP transcription factors in adversity stress,this study screened 27 bZIP genes involved in droughtrehydration stress by analyzing the transcriptome data.After calculation of the correlation coefficients and construction of the co-expression network map,ZmbZIP84(Zm00001d053988)was found to be the core node gene.This gene is located on maize chromosome 4 and has a highly conserved bZIP structural domain.Analysis of the physicochemical properties revealed that the protein coded by ZmbZIP84 is hydrophilic and an unstable protein.A phylogenetic tree was constructed and ZmbZIP84 was most closely related to those in willow millet and sorghum.The quantitative real-time PCR(qRT-PCR)results showed that ZmbZIP84 was expressed in all tissues of maize,with the highest expression in mature roots.Setting up simulated tests such as 20%PEG-6000,42℃high temperature,200 mmol·L^(-1)NaCl and lack of ammonium nitrogen,it was found that the expression of ZmbZIP84 was significantly up-regulated under high temperature,drought,and nitrogen treatments,while the expression was down-regulated under NaCl treatments,which indicated that ZmbZIP84 actively participated in and responded to abiotic stresses.Using CRISPR/Cas9 technology to obtain Arabidopsis thaliana homozygous deletion mutants of ZmbZIP84 gene,it was found that the growth of the mutant plants was severely inhibited,the leaves wilted and even dried to death under high temperature and drought stress treatments,whereas the wild-type plants were less affected,suggesting that knockout of ZmbZIP84 gene reduced the drought and high temperature tolerance.Subcellular localization revealed that the protein encoded by this gene is located in the nucleus.The results of this study laid the foundation for subsequent study on the function of ZmbZIP84 gene and the regulatory mechanism of the downstream target genes,which will provide a reference for breeding new resistant maize varieties.