水稻OsPht1;2和OsPht1;6响应缺磷信号的分子调控机制研究

In order to counteract low phosphorus (P) stress, plants have evolved a series of elaborate adaptive responses. Cis-regulatory element is one of the key factors in transcriptional regulation of P-starvation-responsive genes and maintenance of phosphate (Pi) homeostasis within plants. OsPht1;2 (OsPT2) and OsPht1;6 (OsPT6) are two P-starvation-induced rice Pi transporter genes. In our previous work, the conservation and specificity of OsPT6 promoter in response to P deprivation were demonstrated. Bioinformatic and functional analysis revealed that there are multiple P-starvation-responsive cis-regulatory elements in different regions of OsPT2 and OsPT6 promoters. In the present work, we intend to identify the putative cis-elements conferring P-starvation-induced activity in the promoter regions of OsPT2 and OsPT6 and verify their conservative property by using mutated (a series of truncations and deletions) and in vitro synthesized promoters fusing GUS reporter gene. In addition, EMSA (Electrophoretic Mobility Shift Assay) and yeast one-hybrid system will also be employed to screen and testify the potential transcription factors recognizing these P-deficiency-responsive cis-elements. Accomplishing these studies, it will allow us to elucidate a new conserved molecular regulation mechanism of P-starvation signal transduction.

为了应对低磷环境，植物在进化过程中形成了一系列适应性机制。顺式调控元件是转录水平上调控基因响应缺磷信号，维持植物体内磷素稳态最关键的要素之一。水稻磷酸盐转运蛋白OsPht1;2 (OsPT2) 和OsPht1;6 (OsPT6) 是两个受磷饥饿诱导强烈表达的基因。申请人前期的研究结果证明了OsPT6启动子对缺磷信号的响应具有专一性和物种间的保守性。此外对OsPT2和OsPT6启动子的生物信息学和缺磷响应的初步分析表明，这两个启动子的不同区段存在多个响应缺磷信号的重要顺式调控元件。本项目拟采用外部缺失、内部定点突变以及人工合成启动子融合GUS报告基因等手段分离鉴定出OsPT2和OsPT6启动子上响应缺磷信号的作用元件，并验证这些元件在不同物种间的保守性。在此基础上通过酵母单杂交和EMSA初步筛选与这些调控元件结合的上游转录因子，从而有望揭示出一条新的植物缺磷信号转导分子调控的保守途径。

在水稻磷酸盐转运蛋白Pht1家族中，OsPht1;2（OsPT2）和OsPht1;6（OsPT6）是两个受缺磷诱导强烈表达的基因，对低磷条件下水稻中磷酸盐的吸收和转运至关重要。为了进一步明确OsPT2和OsPT6响应磷饥饿信号的分子作用机制，本项目以OsPT2和OsPT6启动子，特别是OsPT6启动子为研究对象，a．通过外部缺失法，在水稻中证实了OsPT2和OsPT6启动子的不同区域在调节基因响应缺磷信号中扮演着不同的角色，OsPT6启动子上一个138bp的片段存在响应磷饥饿信号的重要顺式调控元件；b．通过内部定点突变法，在烟草中证实了P1BS和W-box顺式调控元件协同作用，参与了OsPT6启动子对磷饥饿信号的响应，其中P1BS元件是其响应缺磷信号必不可少的，距离转录起始位点较近的W-box元件起到增强子的作用，而较远的W-box元件则对OsPT6启动子响应缺磷信号无显著影响；c．通过构建人工合成最小启动子，进一步验证了P1BS和W-box顺式调控元件对缺磷信号的响应能力；d．通过酵母单杂交系统筛选到1个可能与OsPT6启动子缺磷信号响应区段结合的烟草基因NtAHL17-like。上述结果为研究植物磷饥饿信号转导的分子调控机制提供了重要的理论依据，同时也为植物基因工程改良提供了具有使用价值的响应缺磷信号的启动子元件。