调控拟南芥铁离子转运蛋白（AtIRT1) 表达的基因的分离和功能分析

Iron is an essential nutrient for plant growth and development。 Under iron deficiency stress, Arabidopsis roots display an induced expression of ferrous transporter IRT1. FIT is a transcription factor that regulates many iron deficiency induced genes, including IRT1. IRT1 protein is not detectable in fit mutant under iron deficiency, but it is mRNA not completely abolished. This indicates that there are some unknown factors involved in the control of IRT1 expression. Previous report showed that the expression of IRT1 is regulated at transcriptional and posttranscriptional level becuase IRT1 protein accumulated only in the roots of 35S-IRT1 transgenic plants under iron deficiency conditions. Previously, we also demonstrated that FIT interacted with AtbHLH38 or AtbHLH39 to directly confer transcription activation of IRT1 gene. Overexpression of AtbHLH38 or AtbHLH39 with FIT in Arabidopsis changed the protein accumulation of IRT1 from induced to constitutively. This implies that the heterodimer of FIT/bHLH38 or FIT/bHLH39 activate some unknown factors to depress the posttranscription regulation of IRT1 expression. In this project, we will set up a genetic screen to identify and isolate the genes involved in the regulation of IRT1 expression. We constructed a EMS mutant population with the transgenic line ox29/38,which overexpressed FIT and AtbHLH38 and showed tolerance to iron deficiency, and screened mutants which showed chlorotic phenotype as the irt1 mutant. We will isolate the genes and characterize their biological functions to illustrate the molecular regulation mechanisms of IRT1 expression.

铁是植物生长和发育所必需的营养元素之一。低铁胁迫下，模式植物拟南芥的根会大量增加二价铁离子转运蛋白（AtIRT1）的表达，从而增加铁离子的吸收。 AtIRT1基因的表达受转录因子FIT 的调控。缺铁条件下，fit 突变体的AtIRT1表达大量下调，暗示除FIT 外AtIRT1的表达还受其它因子的调控。另外，已有的研究工作表明，AtIRT1 蛋白只在缺铁的野生型根中积累，但我们实验室的工作表明，同时过量FIT和其互作蛋白基因bHLH38能改变AtIRT1 蛋白的表达模式，为组成性表达。这意味着FIT/bHLH38异源二聚体通过激活未知因子的表达同时也启动了植物的缺铁反应。因此，本研究的任务就是以拟南芥野生型和FIT/bHLH38双过量转基因植株为材料，建立EMS 突变体库，在低铁条件下挑选表型与irt1 相似或更严重突变体，鉴定和分离调控IRT1 表达的新基因，并研究其生物学功能。

植物铁离子转运蛋白IRT1在植物耐低铁胁迫中起着重要的作用，关于其转录和转录后调控目前了解还比较少，除了FIT 及其互作蛋白bHLH38和bHLH39 外，还没有其它基因报道。另外，双过量FIT/bHLH38和FIT/bHLH39植株在正常铁供应条件下在根部能积累IRT1 蛋白的机制还没有人研究。因此，本实验室利用野生型拟南芥以及我们实验室所特有的FIT/bHLH38 转基因植株做实验材料，去分离调控IRT1 表达的新基因，填补国际上这块研究空白，完善调控IRT1基因表达的网络。本研究通过对筛选出来的与铁吸收有关的两个突变体lism1 和 lism26 的分析，克隆了两个参与调控IRT1 蛋白表达的两个新基因，并研究了其参与调控的分子机制，其中lism1突变体的工作已在线发表于2014 年 The newphytologist 杂志上，lism26 突变体的工作也正在整理结果当中。本课题通过对两个缺铁调控相关突变体的研究，分离克隆了相应的目的基因，探索了它们的生物学功能和作用机理，对拟南芥铁稳态调控机制有了更深入的理解。