低氮条件下外源C4-pepc基因维持水稻高光合效率的分子机制

The expression of C4-phosphoenolpyruvate carboxylase (pepc) gene can increase nitrogen assimilation of rice under low nitrogen. However, there were few reports about the response of photosynthesis to low nitrogen in C4-pepc transgenic rice recently. The previous study showed that C4-pepc transgenic rice had higher PSII(photosystem II)activity and light energy transfer efficiency, and higher net photosynthetic rate was maintained by increasing key enzymes activities of carbon and nitrogen assimilation under low nitrogen conditions, but the mechanism was still unclear. In the present study, C4-pepc transgenic rice (12th generation) will be used and nontransgenic rice ‘Kitaake’ as control. Changes will be measured in photosynthetic characteristics, the contribution of C4-PEPC to carbon fixation, its related anaplerotic pathway for the TCA cycle, and nitrogen assimilation in flag leaves at filling stage grown under low nitrogen. Analysis will be made in activities of photosynthetic enzymes, products and gene expression of carbon and nitrogen metabolism, to discuss the flow of photosynthetic products to low nitrogen in C4-pepc transgenic rice. The regulation mechanism will be explained in promotion of photosynthesis in C4-pepc transgenic rice by enhancing carbon assimilation and nitrogen use under low nitrogen, in order to provide theoretical support for breeding high photosynthetic efficiency and low nitrogen-tolerant rice.

C4-pepc基因的表达能够提高水稻在低氮条件下的氮素同化能力。但关于转C4-pepc基因水稻对低氮水平的光合响应机理研究较少。前期研究表明，转C4-pepc基因水稻具有较高的PSII活性和光能转化效率且通过提高碳氮关键酶活性来维持低氮条件下高的净光合速率，但其具体机制尚不明确。本项目拟选用第12代转C4-pepc基因水稻为实验材料，采用非转基因受体Kitaake作为对照材料。对材料进行低氮处理，研究灌浆期剑叶在低氮条件下光合功能的变化、C4型PEPC酶对碳同化的贡献程度，以及与PEPC酶相关的TCA辅助反应和氮同化的变化，分析转C4-pepc基因水稻中光合关键酶及其相关碳、氮代谢基因的表达情况和代谢产物的变化，揭示光合产物流向对低氮的响应。阐明转C4-pepc基因水稻增碳促氮的高光合效率的适应调节机理，为培育高光效和低氮耐性水稻提供理论支撑。

本研究选用转C4-pepc基因水稻（PC）为实验材料，以非转基因受体Kitaake（WT）为对照，对材料进行低氮处理，研究其在低氮条件下光合特性及产量、碳氮代谢物含量、关键酶活性和相关基因的表达情况，以阐明转C4-pepc基因对水稻在低氮下增碳促氮的适应调节机制。结果表明：（1）低氮条件下，PC水稻表现出较高的光合效率，其光合系统II和总的光合性能没有受到影响；PC和WT产量的显著差异主要在株高和千粒重上，PC水稻通过转移更多的生物量到籽粒中，以缓解低氮对其产量的影响。（2）与WT水稻相比，低氮条件下，PC水稻对铵态氮的吸收增加，氨基酸含量升高，三羧酸循环（TCA）辅助反应中有机酸含量上升，使得其总可溶性蛋白和地上部生物量提高，能够更好地在低氮环境中生长。（3）低氮可诱导PC水稻TCA辅助反应关键酶（如柠檬酸合酶、异柠檬酸脱氢酶、α-酮戊二酸脱氢酶）和氮素同化关键酶（如硝酸还原酶、谷氨酰胺合成酶、谷氨酸合成酶）活性的增加，促进其碳氮代谢作用，增加其对低氮的耐受性。（4）PC水稻在低氮条件下氮素吸收（AMT1.1、NRT1.1B）、氮素同化（如GS1.1、GS1.2）和TCA辅助反应（NADP-MDH、ICDH1-3）相关基因的表达量增加，以更好地适应低氮胁迫。