ZmPht1;6调控玉米磷吸收转运的分子机制研究

Maize is one of the most important crops, however its production is limited seriously by inorganic phosphate (Pi) starvation. It is urgent to know more on the molecular and biochemical mechanisms involved in Pi uptake and transport in maize. As far now, Pht1s had been mostly researched among all phosphate transporters in plants. And, several Pht1 proteins had been cloned from maize,among them, ZmPht1;6 is inducible to enhance the uptake of Pi by arbuscular mycorrhizal(AM) fungi. However, the molecular mechanism of ZmPht1;6 in medaiting Pi uptake and transport in maize is still kept unknow. In this study, in order to disclose the expression pattern of ZmPht1;6 to AM fungi, the gene expression and the protein level of ZmPht1;6 in maize inbreds which have different sensitive to Pi starvation will analyzed respectively with qRT-PCR, In situ hybridization and Western blotting technologies. Moreover, the subcelluar location of ZmPht1;6 will be investigated by maize protoplast transient expression system and GFP fusion protein subcellular localization. After that, the function of ZmPht1;6 in mediating the Pi uptake and transport will be characterized by Xenopus Oocytes expressing system, functional complementation of yeast mutant, maize and Arabidopsis thaliana overexpressing and RNAi mutant. Finally, ZmPht1;6 will be truncated into several fragments according its conserved domain to discover the key functional domain. Thus, the molecular mechanism of ZmPht1;6 in medaiting Pi uptake and transport in maize could be expected to be revealed, and these results could be anticipated to support the further research in related area and improve the tolerance to Pi starvation in maize by molecular technology.

玉米是最重要的农作物之一，其生产因低磷胁迫被严重限制。生物体磷吸收转运过程主要由磷转运体完成，Pht1是目前高等植物磷转运体中研究最深入的一类。已从玉米中克隆到多个Pht1磷转运，其中ZmPht1;6受丛枝菌根真菌诱导，表达增强，有利于改善玉米对磷的吸收转运。但还未有报道深入阐述ZmPht1;6调控玉米磷吸收转运的机制。本项目拟结合玉米根系ZmPht1;6基因组织表达模式和蛋白表达量，揭示ZmPht1;6对丛枝菌根真菌的应答模式；结合原生质体和GFP亚细胞定位结果揭示ZmPht1;6亚细胞定位情况，然后结合异源表达系统和植物体内正反向功能互补研究结果，明确丛枝菌根真菌诱导下ZmPht1;6对玉米磷吸收转运的调控功能；再定位其中调控磷吸收转运的功能区域；从而系统阐述ZmPht1;6调控玉米磷吸收转运的分子机制。研究结果可为深入理解玉米磷高效机制及玉米耐低磷的分子改良提供科学依据。

项目围绕玉米磷转运体ZmPht1;6，开展玉米磷吸收转运调控分子机制研究。通过比较分析低磷胁迫下多个基因型玉米材料的表型，确定以CML402为研究材料，就低磷胁迫下玉米根系和叶片中基因表达情况开展研究；发现：在根系和叶片中均存在不同数量的差异表达基因；而且在低磷胁迫下ZmPht1;6在根系和叶片中表达量均增加。进一步分析发现低磷胁迫抑制玉米光合能力，另一方面却诱导花青素积累、GAD酶活性增加和GABA积累。外源GABA诱导多个抗氧化酶活性增加，促进脯氨酸积累，而且诱导ZmPht1;1-ZmPht1;6表达量增加和玉米磷吸收；GABA合成抑制剂处理则作用相反。推测ZmPht1;1-ZmPht1;6等磷转运体在调控玉米磷吸收和转运过程中受到GABA信号调控。同时研究发现ZmPht1;6在核膜和其他小细胞器膜有分布。利用非洲爪蟾卵母细胞表达系统证实ZmPht1;6响应高浓度磷即10mM NaH2PO4，暗示其可能具有低亲和磷转运功能；综合低磷胁迫下玉米基因表达情况分析中的发现，推测ZmPht1;6可能兼具高亲和和低亲和磷转运功能。利用生物软件分析发现ZmPht1;6蛋白结构中有大量的α螺旋，这些α螺旋形成了一个中心通道，该通道可能与其转运功能相关；而且其中319-329氨基酸区间的AFYSQNLTQKD与磷酸根离子对接，可能是其底物结合关键区域。本研究结果为综合阐述 ZmPht1;6 调控玉米磷吸收转运的分子机制和深入理解玉米磷高效机制提供科学依据。