高钙启动能量补偿增强转C4光合基因水稻光合效率的机理

The internatinal efforts are underway to engineeer C4 photosynthesis into C3 crops,which would be an important and new scientific strategy for the next green revolution challenges.The energy mechanism in the C4 photosynthetic pathway is one of the important topic on C4 rice.However, the energy mechanism on the C4 photosynthetic pathway in transgenic plants was still very still very less,which limited the progress on C4 rice. The project intends to elucitate the mechanism of energy metablism mediated by calcium in transgenic rice plants with high level expression of C4 genes .At first,the special two-way-synthesis of calcium induced to increase under all kinds of environmental factors,such as high light intensity conditions by determination the contents of Ca2+.The molecular basis was studied by analsing by both the key bases of the promotor in C4 pepc gene and the relationship between C4 gene and the transcription factor, Dnof1 by calcium,which would be helpful to understand the molecular basis of the energy metabalism in transgenic rice plants with over-expression C4 photosynthetic genes.The sites of calcium releasing and transferring fuction were determined by all kinds of methods on calcium determination such as calcium trace,calcium tags and calcium positioning.Next,the regulation of calcium on the signalling pathway mediated by PA produced by the phospholipase D pathway were then studied for the effects of phosphorylation produced ATP(Photophosphorylation and oxidative phosphorylation),therefore induced the production of ATP and produced the photosynthetic acclimation or adjustment mechanism. Finally ,the biological and physiological roles on the energy pathway mediated by calcium on enhancing the photosynthesis in C4 photosynthesis gene transgenic rice plants were proved by different levels such as the plant, cell, photosynthesis system and genes. The project will be helpful to understand more about the theory of the C4 rice and will provide the basis for the use of C4 gene transgenic rice plants on the genetically modified rice breeding for super-high-yield in practice.

构建C4稻是引领下一次绿色革命的一个重要科学策略，其中转C4光合基因水稻能量代谢机制是构建C4稻的重要方面，但有关研究还很分散，在一定程度上限制了C4稻的研究。 本项目拟以高表达的转C4光合基因水稻为材料，以内源钙为中心，首先采用钙定量和定性的方法相结合，研究逆境下（高光强）内源钙的独特双向产生机制，再通过分子生物学和生物信息学方法，研究钙介导C4光合基因启动子与其结合的转录因子Dof1互作的分子基础，并通过钙示踪和荧光定位等方法，研究钙信号的传递规律，并以磷脂酶Ｄ合成的磷脂酸（PA）和钙信号互作，研究其参与促进能量产生的磷酸化过程（光合磷酸化和氧化磷酸化），启动ATP补偿，引发其光合机构的适应调节机制，期望从植株、细胞、光合机构和基因等水平上，阐明钙介导能量代谢对转C4光合基因水稻光合作用增强的机理。 本研究将丰富构建C4稻理论，并在实践上加快转C4光合基因水稻在超高产育种中的应用。

C4-稻的培育是植物科学家和作物育种学家共同的目标。本项目以稳定高表达的转C4-光合基因水稻为研究对象,分别以植株和悬浮细胞为研究模式,重点从钙作为本项目的着眼点.通过外源施加钙以及内源钙抑制剂(螯合剂和钙通道抑制剂)正反两方面,研究钙对高表达的转C4-光合基因水稻光合特性以及产量的影响,并在细胞水平,进一步从干旱逆境为处理条件,分别从信号传导,基因的响应以及生理适应机制。研究结果揭示了内源钙离子参与高表达转C4-PEPC基因水稻的干旱响应作用机制：即高表达的转C4-光合基因水稻在感受干旱胁迫时,通过快速动员和感受内源Ca2+(calcium ion)水平,协调H2O2(hydrogen peroxide)、NO(nitric oxide)和ATP(adenosine triphosphate)信号,在DNA水平上,通过去甲基化方式增强了导入的水稻C4-PEPC全基因的表达,并在翻译后水平,通过磷酸化方式,增加了PEPC酶活性,从而表现了较高的干旱逆境的响应和耐受能力，进一步的RNA-Seq和RT-PCR结果显示了多种激酶基因参与了转基因水稻的干旱响应，其中蔗糖非发酵1 (SNF1)相关蛋白激酶家族(SnRKs)如SnRK1A、OsK24和OsK35等基因中诱导增加,提示了SnRK1亚家族基因在转C4-光合基因水稻高光合能力和应对干旱逆境的平衡作用。本项目提出了表观遗传方式-DNA甲基化也参与了该材料耐旱的新机制,相关的研究结果为丰富植物耐旱的分子机理提供新线索。本项目发表了标注论文20篇,其中包括SCI论文收录的8篇论文。在学术交流方面：本项目执行期间,参加了17次学术会议,包括4次国际学术大会,6次全国性学术会议,2次地区间学术会议以及5次省级学术交流,并在3次地区间学术会议作分组报告,获得了5次省级学术会议的优秀论文报告奖。在人才培养方面：本项目派出了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8位研究生全程参与了本项目的实施，其中1位获得博士学位以及6位获得了硕士学位，提升了本项目参加人员的科研能力。