铁信号感应器关键位点的鉴定和泛素化调控

The iron content is very low in rice grains whereas relatively abundant in bran organs. The reasons for grain “anemia” are that one is the bran cells cannot discharged abundant iron into bran cells through excytosis. And another is grain cells cannot accept (uptake) the iron from the supplier-bran. However, researches about which one acts as the iron-sensor and how the iron-sensor controls the Fe-transporter on-off through regulation in these cells is still sparse. Our team focuses on both of these issues and aims at providing theoretical basis on improving grains iron nutrition. The research programmes are list as following:① Molecular structure analysis of iron-regulated transporter IRT1s in subcellular level and transcription factor IDEF1 in transcriptional level, including construction of H1-H5 sites mutants which are supposed to be metal acceptor and lysine (K) sites mutants which are probably involved in protein ubiquitination.② Establishing transgenic yeast and rice suspension cell lines, testing the function of iron-sensor through immobilized metal-chelated affinity chromatography (IMAC), and screening the key functional amino acids involved in sense iron signal through functional complementary experiment, immunofluorescence laser scanning confocal microscope, Non-invasive Micro-test Technique, ICP-MS.③ Screening the partners with the bait of MxIRT1 using ameliorative Co-IP-TAP and split ubiquitin yeast two-hybrid system through iron-improved transgenic rice and related mutants and identifying the function of these partner to validate the regulation of ubiquitination on iron-sensor.In a word, it will provide the theoretical basis for rice grain iron enrichment of the breeding goal.

水稻籽粒铁含量很低,但糠壳等含铁充足。分析其籽粒“贫血”的原因,一是糠壳等细胞不能将充足铁外排到籽粒中,二是籽粒细胞不能有效接受(吸收)营养细胞提供的铁。在这些细胞中，谁是铁的感应器？它又是如何被调控的?本课题聚焦在本领域最热点的两个问题进行深入研究，为改善水稻籽粒铁营养提供理论依据。其研究方案为:①对铁转运体IRT1s和转录因子IEDF1做更深入的分子结构解剖，构建可能接受金属离子的H和泛素化K的突变载体; 细胞水平研究关键氨基酸对定位和功能的影响；②转化酵母和水稻悬浮细胞系，利用固定化金属离子亲和层析（IMAC）的技术鉴定铁感应器的功能；利用异源互补，共聚焦观察，非损伤测试，ICP-MS，筛选感应铁信号的关键氨基酸；③利用已能提高铁含量的MxIRT1转基因水稻,拟南芥及相关突变体，采用改良的Co-IP-TAP等，筛选铁感应器和泛素化的复合体，观察表型，验证泛素化在铁感应器的调控作用。

铁是植物人体营养的必要元素，铁缺乏已成为影响植物生长和人类健康的因素之一，特别是以水稻为主要粮食的发展中国家。为更深入解析铁感应机理，从铁吸收高效苹果中克隆的MxIRT1入手研究其感应铁及调控机制。.1.生物强化解决水稻铁缺乏.构建MxIRT1正义反义转基因水稻稳定系。反株系变小且叶片偏黄。正MxIRT1高表达使其更耐受缺铁锌且籽粒中铁锌含量高三倍。比较AtIRT1、OsIRT1、MxIRT1转运镉吸收，MxIRT1最低。环境中铁能抑制MxIRT1对镉的吸收。说明MxIRT1是可用于铁锌生物强化的很好基因资源。.2.铁感应器（Fe-sensor）或铁受体（transporter-recepter）.2.1 对 loop 环富组氨酸基序（HRM） H184、H186、H188、H190、H192 进行点突变。固定化金属离子亲和层析（IMAC）证明 loop 环能够直接螯合 Fe2+，其中H184，H192 是关键位点。转基因拟南芥 MxIRT1H192A 和 MxIRT1H184/192A 与 irt1缺失突变体表型相似，矮小、叶片萎黄，叶绿素低，铁镉低，Cd2+外排。.2.2 脂筏结构域（CRAC）L262、Y266、K270突变，叶绿素值，加入脂筏染料Filipin的共聚焦显微镜观察，Cd2+流速和外排，植株表型等，MxIRT1Y266A与野生型差异极显著，证明MxIRT1Y266A是感应铁而影响质膜脂筏定位和离子转运的关键位点。.3.Loop环上K165及K196泛素化.K165A 突变对 MxIRT1 蛋白定位生理功能表型无影响。但K196A 突变和 K165A/K196A 双突变则影响很大。Co-IP 验证是失去了多点单泛素化而影响蛋白的 turnover。还对早期缺铁CSN6表达量的变化以及对IDEF1蛋白泛素化的调控进行了研究。.4.通过罗丹明 B 与乙酰丙酮缩合反应成果研发了灵敏度高达 10−6 mol/L 的荧光型 Fe(Ⅲ)感应剂 RA。