玉米ZmSNAC1基因调控植物耐旱的机制研究

NAC transcription factors are plant-specific regulators that have multiple biological functions including stress adaptation and tolerance, plant development, morphological formation and hormone regulation. Recently, NAC transcription factors have received more attention for the great achievements in research on the functions of NAC members in model plants, especially in rice and Arabidopsis. However, the regulating mechanisms of plant stress tolerance mediated by NAC transcription factors, and the functional analysis of maize NAC members in response to abiotic stresses have not been characterized. Drought is one of the major environmental stresses that adversely affect the growth and productivity of maize. At present, the roles and regulatory mechanisms of maize NAC genes in plant drought stress tolerance were still unknown. In this study, we will explore the upstream regulators and interacting protein of ZmSNAC1, and analyze its expression pattern under various stress treatments including drought and ABA. The key region of ZmSNAC1 that involved in protein interaction will be also identified. We will further employ RNA-Seq analysis to identify the differences in gene expression between the ZmSNAC1 overexpression lines, the RNAi lines and the wild type lines under drought stress conditions. In addition, we will characterize the different expressed genes for its important role in stress tolerance. Together, this work will contribute to analyze the drought tolerance mechanism of ZmSNAC1 gene in maize and will be beneficial in molecular breeding to improve crop stress tolerance.

NAC转录因子是植物特有的具有多种生物学功能的转录调控因子，因其在抵抗逆境、调节植物生长发育等方面发挥的重要作用，近年来受到研究者的广泛关注，特别在模式植物如拟南芥中NAC家族成员的研究已取得了丰硕成果，但NAC基因的抗逆调控机制及其在重要粮食作物玉米中与抗逆相关的功能研究较少。干旱是影响我国玉米生产、造成粮食减产的重要限制因素之一。目前，玉米中NAC基因耐旱功能的研究仍处于起步阶段，玉米NAC基因调控其耐旱性的作用机制尚不清楚。本研究将进一步明确ZmSNAC1在玉米中的耐旱生物学功能，筛选并鉴定ZmSNAC1上游调控因子和互作蛋白，分析其在干旱和ABA胁迫下的诱导表达模式；明确ZmSNAC1转录调控机制和蛋白互作关键氨基酸序列；分析干旱胁迫下超表达ZmSNAC1株系、RNAi和对照株系的差异表达基因，鉴定其靶基因及生物学功能，探讨ZmSNAC1介导的耐旱分子调控机制，创制耐旱玉米新种质。

NAC转录因子在抵抗逆境、调节植物生长发育等方面发挥重要作用，目前在模式植物中已取得了丰硕研究成果，但NAC基因的抗逆调控机制及其在玉米中与抗逆相关的功能尚不明确。本研究将进一步明确ZmSNAC1在玉米中的耐旱生物学功能，采用酵母杂交、表达分析、RNA-seq、生物信息学分析等技术手段筛选并鉴定出ZmSNAC1上游调控因子、互作蛋白和下游靶标基因，分析调控基因在干旱胁迫下的诱导表达模式及生物学功能，探讨ZmSNAC1介导的耐旱分子调控机制，创制耐旱玉米新种质。本项目主要结果如下：.（1）构建玉米cDNA文库，采用酵母单杂交实验方法，以ZmSNAC1启动子DNA非自激活区域为诱饵筛选得到ZmSNAC1上游调控因子编码基因，共获得基因No序列82条，Go分析结果表明其中约35%和50%的基因分别参与应答非生物逆境胁迫和外界刺激。.（2）以ZmSNAC1非自激活区为诱饵，采用酵母双杂交实验方法，筛选并鉴定出ZmSNAC1互作蛋白编码基因，经一对一互作验证后共得到包含2个NAC基因在内的18条阳性非冗余基因序列；对其中15个ZmSNAC1互作蛋白编码基因在干旱胁迫和复水处理下的诱导表达模式进行分析，结果显示XP_008644394与ZmSNAC1基因表达模式一致；XP_008644394的耐旱功能分析结果表明XP_008644394过表达转基因拟南芥耐旱性较野生型植株显著提高，由此推测其可能与ZmSNAC1互作共同参与调控玉米耐旱过程。.（3）利用玉米幼胚转化法转化共获得17个转基因玉米阳性自交系单株，创制玉米抗旱种质材料1份，明确了ZmSNAC1过表达转基因株系苗期的耐旱生物学功能；酵母单杂交实验分析结果显示，玉米ZmSNAC1与含有NAC核心结合元件序列互作；RNA-seq分析干旱胁迫和对照处理下ZmSNAC1靶基因在ZmSNAC1转基因玉米超表达株系和对照株系中的表达量变化，结果表明ZmSNAC1通过调控RD29A、ERD1等逆境胁迫应答靶基因的表达来提高转基因植株耐旱性；最终解析以ZmSNAC1为中心的干旱胁迫应答信号转导通路。