小麦TaZFP15调控植株应答低磷分子网络和生化途径研究

As important members in the low-Pi stress signaling transduction system, distinct transcription factors play critical roles in it by fine-regulating the expression of the downstream genes. Our previous results clearly indicated that TaZFP15, a zinc-finger type transcription factor gene in wheat, is involved in responding to low-Pi stress and up-regulation of this transcription factor gene could significantly improve the capacities of plant growth, phosphorus acquisition, and phosphorus utilization under deficient-Pi condition. In this study, we will further identify the subcellular location of TaZFP15 based on in situ hybridization and clarify the transcriptional regulation mechanism regulated by TaZFP15 via analyzing expression of fusions constructed with truncated promoter fragments and reporter gene GUS. Using the T3 transgenic wheat lines we have generated, we will systematically identify the differentially expressed genes, proteins, and metabolic related substances that regulated by TaZFP15 based on techniques of gene chip and hybridization, protein 2-DE isolation and corresponding MS identification, and LS-MS and GS-MS, respectively. Based on above research, we will dissect the molecular network regulated by TaZFP15 and systematically determine the biochemical metabolic pathway to respond and withstand the starvation-Pi stress in plants with overexpression of TaZFP15. Taken together, the current project will profoundly bring to light the molecular mechanism on response and resistance of low-Pi stress in wheat from the scope of transcriptional regulation. The results and achievements will provide the theoretically basis for breeding cultivars of wheat and other related species with high-P use efficiency.

植株体内存在复杂且精细的应答低磷逆境转导通路，特定转录因子作为通路重要成员在调控下游低磷应答基因的表达中发挥重要作用。申请者前期研究表明，小麦C2H2型锌指蛋白类（ZFP）转录因子基因TaZFP15对低磷逆境产生明显应答，在增强植株抵御低磷胁迫中发挥着重要功能。本项研究拟在前期工作基础上，进一步鉴定TaZFP15的亚细胞定位特征，通过原位杂交和启动子不同长度片段驱动报告基因（GUS）表达研究，揭示TaZFP15应答低磷的转录调控机制。以目前已获得的超表达TaZFP15的转基因植株为材料，利用基因芯片技术、蛋白质组学技术和代谢谱分析技术，系统鉴定TaZFP15调控的下游基因、蛋白和代谢物，解析该小麦转录因子调控的分子网络，阐明其调控植株应答和抵御低磷逆境的生化代谢途径。本项研究将从转录调控角度，深刻揭示小麦应答和抵御低磷逆境的分子机理，为今后创制磷高效小麦及其他作物种质（品种）提供理论依据。

1、项目背景.提高作物对磷肥的吸收和利用效率，对于节省磷肥投入成本、促进不可再生磷素资源可持续利用具有重要的实践价值。本项目针对迄今有关重要转录因子基因介导小麦应答低磷逆境分子机制研究尚少的现状，以对低磷逆境明显应答的小麦ZFP型转录因子基因TaZFP15为基础，系统开展该基因调控植株抵御低磷逆境分子机理。.2、项目主要研究内容.采用现代分子生物学技术，系统研究TaZFP15编码蛋白的亚细胞定位和应答低磷逆境的转录特征。以超表达TaZFP15的典型转基因系植株和对照植株为材料，系统研究该基因特异调控的转录谱、蛋白质图谱和代谢谱，鉴定其特异调控的基因、蛋白质和代谢物质，建立该基因介导小麦应答和抵御低磷逆境的生化代谢途径。.3、项目重要结果. （1）采用激光共聚焦检测技术，鉴定了TaZFP15的亚细胞定位，发现其定位在细胞核内；（2）原位杂交和TaZFP15-GUS表达检测结果表明，TaZFP15在根系表皮、周边细胞及维管组织优势表达；（3）采用基因芯片、蛋白质组学和GC-MS分别检测了TaZFP15特异调控的基因、蛋白质和代谢物，发现该基因调控129个基因和22个蛋白质的特异表达，影响18种代谢物的含量。表明TaZFP15通过调控众多基因、蛋白表达，影响植株低磷逆境下的生化代谢过程；（4）对TaZFP15特异调控的3个耐低磷关键基因，包括磷转运蛋白基因TaPT2、焦液泡焦磷酸酶基因TaVP和锌指型基因TaZAT8分子特征及介导植株抵御低磷逆境功能进行了研究，在低磷条件下，遗传转化上述基因可显著增强植株磷素吸收、改善植株生长、干物质积累和细胞保护功能；（5）TaZFP15通过调控其下游与低磷抵御重要基因TaPT2、TaVP和TaZAT8等的转录，介导植株中低磷信号转导，建立植株转录因子-下游防御基因互作的抵御低磷逆境途径。.4、关键数据. 依托本项目研究，课题组取得了包括TaZFP15亚细胞定位、TaZFP15转录调控、特异调控的转录谱、蛋白谱和代谢谱，以及TaZFP15调控重要下游基因分子特征及功能等相关数据。.5、本项目科学意义. 本项目鉴定了小麦种属中介导低磷逆境的重要转录因子基因TaZFP15，构建了TaZFP15与其调控下游基因介导植株抵御低磷逆境的生化途径。该途径成员对于今后鉴定小麦种质磷效率及指导小麦磷高效遗传改良具有重要指导价值。