玉米吸收铁的作用机理研究

This study focus on the important scientific issues including iron absorption, transportation, distribution, and the regulating mechanism, as well as the essential practical problems of improving iron content in maize grain. We plan to research the function of the related genes (ZmH+ATPase、ZmFRO、ZmIRT1) and transcript factor (ZmFIT), which participate in Fe uptake strategy I. Then, the influence of molecular characteristics on the physiological functions of selected genes can be unraveled by investigating the phenotypes of overexpression and CRISPR transgenic maize plants. Moreover, the roles of the genes in maintaining the homeostasis of iron in maize can be clarified by analyzing the expression of the key genes participated in the uptake and transportation of iron and determining the iron contents of the transgenic and wild-type plants. With the aim of screening those proteins that interacted with ZmFIT, a yeast two-hybrid screening was performed by constructing the cDNA library. Positive colonies can be further confirmed using co-immunoprecipitation (Co-IP) analysis and bimolecular fluorescence complementation (BiFC) assays. The role of the iron uptake strategy I can be clarified by investigating the ZmFIT participated in the expression regulatory networks and the regulate role of ZmFIT in the expression of ZmFRO and ZmIRT1. Our results may provide candidate genes with independent intellectual property and basic theory for efficient breeding of maize varieties with high iron contents.

本研究基于玉米对铁吸收、运输方式、分布规律和调节机制等重要的科学问题以及提高玉米籽粒中铁含量的重要实际问题，拟选择参与机制I铁吸收、转运的相关基因（ZmH+ATPase、ZmFRO、ZmIRT1）以及参与调节基因表达的转录因子（ZmFIT）进行研究。构建过表达及CRISPR载体并转化玉米，通过测定转基因与野生型植株中铁含量以及分析转基因植株中参与铁吸收、转运等其它基因表达水平的变化，揭示ZmH+ATPase、ZmFRO、ZmIRT1、ZmFIT基因的功能；构建cDNA文库通过酵母双杂交筛选ZmFIT的互作蛋白，通过Co-IP及BiFC进一步验证互作蛋白；研究ZmFIT参与的表达调控网络及其对ZmFRO与ZmIRT1的调节作用，探明玉米中铁的吸收机制I的机理。为铁高效转基因育种提供具有自主知识产权的候选基因，为铁高效育种奠定理论基础。

铁是动植物生长发育必需的微量营养元素。植物缺铁可能造成生长抑制和产量降低，人类缺铁会导致贫血。全世界约三分之一的人群面临缺铁的威胁，且人类主要依赖从植物中获取铁。因此，研究植物吸收及运输铁的机制对于植物生长发育和人类健康均具有重要意义。本研究选择参与机制I铁吸收、转运的相关基因（ZmFRO2、ZmIRT1、ZmIRT2）以及参与调节基因表达的转录因子（ZmFIT）进行研究。构建过表达及RNAi载体转化玉米，通过测定转基因与野生型植株及种子中铁含量的变化及分析转基因植株中参与铁吸收、转运等关键基因表达水平的变化，初步揭示了ZmFRO2、ZmIRT1、ZmIRT2基因的功能。构建玉米cDNA文库通过酵母双杂交筛选出与ZmFIT 互作的ZmbHLH101蛋白，通过BiFC进行了互作验证；通过对ZmFIT过表达转基因材料中ZmIRT1、ZmIRT2及ZmYS1的表达变化，推测ZmFIT可能与ZmbHLH101互作来调控ZmIRT1、ZmIRT2及ZmYS1的表达，这三个基因又通过共同协作来保持玉米体内铁离子的平衡。以上研究可为铁高效转基因育种提供ZmIRT1和ZmIRT2两个功能基因，为玉米铁的吸收机制研究奠定理论基础。