杨树PtPEPCK1调控碳氮平衡的分子机理研究

In this project, 84K poplar was used as the experimental material for overexpression and RNAi inhibition of expression of PtPEPCK1 gene. The results of differential pathway analysis in transgenic plants showed that overexpression of PtPEPCK1 gene had a great effect on the TCA cycle while RNAi inhibition showed its involvement in lateral branch of gluconeogenesis rather than complete gluconeogenesis. The subsequent experiments focused on the biological significance of this gene on the regulation mechanism of carbon skeleton. With statistical analysis of growth phenotypes as the starting point, we summarized the changes of the corresponding substances at transcriptional and metabolic levels and explored on trends of correlation among different pathways as obtained from whole transgenic plants at mRNA and final metabolite levels. While combining protein localization, interaction, and carbon-nitrogen stress experiments, the PtPEPCK1 perturbed core metabolic pathways were revealed from multiple perspectives explaining the biological significance of how the gene affects the reduction and assimilation of nitrogen through regulation of the carbon skeleton. Poplar is a perennial woody plant, and its carbon and nitrogen network mechanism and related major scientific problems are not revealed by annual herbs. The analysis of carbon and nitrogen balance regulation mechanism of PtPEPCK1 in poplar has great theoretical and practical significance for the growth and development of woody plants.

本项目以84K杨树为实验材料，将PtPEPCK1基因超量表达和RNAi抑制表达，差异通路分析结果显示过表达该基因对TCA循环产生较严重的影响，RNAi抑制表达在TCA循环局部产生回补，转PtPEPCK1基因参与糖异生旁路代谢而非完整的糖异生过程。后续的研究侧重该基因对碳流调控的生物学意义解析，以生长表型统计分析结果为切入点，总结其在转录和代谢层面相应物质的变化规律，加强各通路间的关联分析，从总体上获得转基因植株在mRNA水平和最终代谢产物水平的整体特征和关键节点。结合蛋白定位、互作、氮胁迫等实验，多方面、多角度揭示PtPEPCK1扰动的核心代谢通路，诠释该基因如何通过调控碳流变化，对氮代谢的还原、同化产生影响。杨树是多年生的木本植物，其碳氮网络机制及相关重大的科学问题是1年生草本无法揭示的。PtPEPCK1在杨树中的碳氮平衡调控机制的解析对木本植物的生长发育具有重大的理论及实践意义

磷酸烯醇式丙酮酸羧化激酶是处于代谢交叉口的关键酶，本身又是一个双功能酶，可行使其脱羧或羧化活性对其底物进行调控，进而直接或间接地参与生物过程。涉及到的生物学过程和通路较多，它的变化必然引起相应代谢通路、机制相应的发生变化，甚至会直接或间接引起植物体内的并发机制的产生。.本项目前期对PtPEPCK1进行转基因杨树研究，探讨其在转录和代谢层面相应物质的变化，深入挖掘转录组和代谢组各个通路间物质变化的趋势，多方面、多角度揭示PtPEPCK1基因如何扰动核心代谢通路。拟通过超量表达和RNAi抑制表达PtPEPCK1，对影响到的核心代谢通路碳流、氮流变化趋势进行差异分析，并对转基因植株进行mRNA水平和代谢物水平的关联分析，精准定位了PtPEPCK1如何调控碳骨架核心位点。超量表达PtPEPCK1使TCA循环、丙酮酸代谢异常，碳流从丙酮酸过多的流入乙酰辅酶A，乙酰辅酶A引碳流入TCA循环，而丙酮酸、乙酰辅酶A、柠檬酸、α-酮戊二酸等都是谷氨酸/谷氨酰胺的生物合成前体，促使碳流进一步流入Glu-Orn循环，导致鸟氨酸循环及其前体物质全部上调（5-49倍）。RNAi抑制表达PtPEPCK1产生了苹果酸向草酰乙酸、琥珀酸向富马酸方向的回补反应，这些物质是天门冬氨酸合成前体，使植物开启苹果酸-天冬氨酸穿梭和转氨作用。本项目精准定位了PtPEPCK1如何调控整体和局部碳流走向，找出PtPEPCK1调控碳骨架的核心通路和关键节点，从而获得PtPEPCK1基因引起通路变化的整体特征，进而阐明PtPEPCK1调控碳氮平衡的分子机理。