高光效转C4光合基因水稻SnRK调节糖分流增强耐旱性的机制解析

The international efforts are underway to engineer C4 photosynthesis into C3 crops, which would be one of the most important and novel scientific strategies for the next green revolution challenge. The international research was conducted to reveal the regulatory mechanisms underlying C4 photosynthesis at both whole-genome and individual gene duplication levels using the next generation sequencing, phylogenetic analysis and mathematical models. In order to apply ‘C4 rice’ to super-high-yield breeding as soon as possible, the domestic scientists created a lot of useful lines by transferring some key C4 photosynthetic genes into C3 crops, which could give these C3 crops higher photosynthetic ability and stronger stress tolerances. However, the assimilate mechanisms underlying C4 photosynthetic pathway in transgenic plants were still very indeterminate, which limited the further applications of these ‘C4 rice’ in super-high-yield breeding. .This project intends to elucidate the mechanism of the photosynthesis in C4 gene transgenic rice plant which conducts assimilating process by SnRK. Using the transgenic C4 rice in which the photosynthetic genes were highly expressed as materials and aiming at the scientific topic of sugar transportation and distribution, firstly an analysis and identification of the specific SnRK gene under drought and sugar starvation were conducted to verify their molecular functions as the bridges between external factor of drought and PA on plasma membrane. Furthermore, the signal integrations in C4-pepc promoter response of both hexokinase signal and drought stress signal transductions were identified on the basis of publicly available sets of RNA-Seq data. Then, through the method of epigenetics, the molecular mechanisms of how SnRK kinase mediated transmembrane PA participating in the specific regulatory regions of C4-pepc and enzyme activations were studied further. Finally, combining with isotope tracer, fluorescence and metabonomic methods, sugar mechanisms by the model of CO2 signal-SNRK-C4-PEPC activation were studied, which would draw the picture of how SnRK participating in the sugar metabolism by increasing both the photosynthetic efficiency and drought tolerance in transgenic rice plants with high expression level of C4-gene. .This project will contribute to further and deeper understandings about the theory of the ‘C4 rice’ and will open a new field for the use of C4 gene transgenic plants in the genetically modified rice breeding for super-high-yield in practice.

构建C4水稻是引领下一次绿色革命挑战的一个重要科学策略。国际上现多用二代测序和数学模型在全基因组水平分析C4光合途径的基因表达，转录和翻译等机理，国内科学家则创制了高光效且高表达的转C4光合基因水稻材料，但有关转基因材料高光效且耐逆的机制研究还很分散。.本项目以高表达的转C4光合基因水稻为材料，针对糖配置的科学问题，首先分析和鉴定对干旱和糖饥饿反应的SnRK基因家族，验证内源钙和磷脂酸（PA）介导SnRK基因对CO2信号的感受；然后通过表观遗传学的方法，鉴定己糖激酶信号和干旱胁迫信号传导的共享元件，明确CO2强化信号下， SnRK激酶介导PA参与C4-PEPC酶的活化机制，最后通过同位素示踪、荧光和组学等方法，研究糖的分流特征及定位， 解析CO2信号-SnRK-C4-PEPC酶模式介导糖的运输，参与高表达转C4-pepc基因水稻高光效和耐旱的作用模式，为将来“C4稻”的培育开辟新领域。

本项目以糖信号为重点，从糖信号元件SnRKs 家族为核心，研究了在干旱条件下（自然干旱和模拟干旱）下，高表达转玉米C4-PEPC基因水稻（以下简称PC）的SnRKs介导的PEPC酶的调控机制。主要研究结果如下：在干旱逆境下，外源导入C4-pepc的高表达，增加了PEPC酶活性以及HCO3-水平，通过改变HCO3-/CO2信号负调节的气孔运动，从而维持了干旱胁迫下PC植株稳定的光合能力；与此同时，与未转基因野生型（以下简称WT），PC体内显示了内源糖浓度显著差异，在干旱逆境下，PC一方面通过不同水平糖组分激活SnRK介导糖信号系统，增强了外界逆境信号感受，另一方面通过加强的细胞壁转化酶活性，降解细胞壁，增加膜的通透性，导致糖分子、钙和NO等第二信号分子的转运加强，诱导了跨膜的蛋白激酶SnRKs家族基因的时空表达, 激活其下游的干旱响应机制。SnRKs家族基因时空表达的具体机制为：细胞水平上诱导SnRK1增强氧化磷酸化以增加细胞的能量水平、蔗糖介导SnRK1s和SnRK2s调节C3和C4型的PEPC基因增强了干旱逆境下的发芽能力、葡萄糖通过NO负调节Ca2+-CBL-CIPK-SnRK3s信号模块参与了气孔运动、蔗糖通过Ca2+-SnRK3s的正调节以及ABA-SnRK2s负调节增强了植株的花青素代谢，最终表现耐旱能力增强。本项目完成了计划的研究内容，发表了项目标注论文15篇, 其中包括SCI收录的7篇;本项目执行期间,参加了17次国际,全国和省级等各类专业性学术会议或论坛,发表会议论文18篇，并有1人做分组主题报告，5人次作分组交流； 2017年派出了1名青年科技人员赴美国加州大学洛杉矶分校进行为期一年的合作研究，并资助了10位硕士研究生全程参与了本项目的实施，其中6位硕士研究生分别获得了硕士学位。本项目按预算和经费使用的要求，规范地使用项目经费。