SlPGR5/PGRL1介导番茄响应低夜温的环式电子传递光保护机制

The protected tomato (Solanum lycopersicum L.)in winter and spring seasons cultivation easily suffer from low night temperature(LNT),which result in photoinhibition, produce excess light energy, then destroy photosynthetic apparatus. Cyclic electron flow (CEF) can protect the photosynthetic apparatus suffering photodamaging,Which is mediated the proton gradient regulatory proteins composite (PGR5/PGRL1),nevertheless,the mechanism of action is still dimness. We obtain transgenic plants through the CRISPR/Cas9 knock out and overexpression techniques, which are subjected to 6℃ LNT treatment. On this basis,first,we realize the characteristic of the gene and protein expression of SlPGR5 and SlPGRL1 in space and time. Second, we analysis CEF rate and accumulation and distribution of ROS in a bid to clear SlPGR5 and SlPGRL1 role in CEF and accumulation of ROS.Then, to investigate the photoprotection effect of the SlPGR5/PGRL1 mediated CEF by analysis of photosystem activity using GFS-3000 and DUAL-PAM-100 synchronous measurement system and green fluorescence imaging system. Finally,in order to clarify photoprotection approach of SlPGR5/PGRL1 mediated CEF by analyzing the change of the heat dissipation,D1 protein turnover and differential protein in thylakoid membrane.We clear ultimately the photoprotection mechanism of CEF in tomato mediated by SlPGR5/PGRL1 in response to LNT, which will provide a new proposal for the regulation photosynthesis in tomato under LNT.

设施番茄生产中常出现低夜温导致的光合抑制，从而产生过剩光能破坏光合机构。环式电子流（CEF）可保护光合机构免受光破坏，而这一过程受质子梯度调节蛋白复合体（PGR5/PGRL1）的介导，但其介导机制尚不清楚。本项目拟在超表达和敲除所获得的转基因植株基础上，经6℃低夜温胁迫后，采用qRT-PCR等技术，明确SlPGR5和SlPGRL1基因和蛋白时空表达；分析CEF和活性氧积累与分布，明确SlPGR5和SlPGRL1在CEF运转和活性氧积累中作用；采用GFS-3000和DUAL-PAM-100同步测量及叶绿素荧光成像系统分析光系统活性，明确SlPGR5/PGRL1介导CEF的光保护作用；分析热耗散、D1蛋白周转及类囊体膜差异蛋白的变化，明确SlPGR5/PGRL1介导CEF的光保护途径。最终明确SlPGR5/PGRL1介导番茄响应低夜温的CEF光保护机制，为调控低夜温下番茄的光合作用提供新途径。

我国北方地区日光温室越冬生产时低夜温现象普遍存在，低夜温导致光抑制，引起叶片光合效率的降低。植物叶片具有多种保护策略，其中围绕PSI的环式电子传递(CEF)对于低温逆境下光保护十分重要，尤其是在C3植物中比较重要的PGR5/PGRL1途径。本研究首先通过获得番茄PGR5、PGRL1的基因编辑材料，以及过表达和RNAi的T1代材料，分析了PGR5/PGRL1的功能以及对低夜温下番茄叶片光保护的调节作用。主要结果如下：①利用Crispr/cas9基因编辑技术获得番茄PGR5和PGRL1突变体植株，并获得PGR5/PGRL1过表达和RNAi沉默T1代材料。PGR5/PGRL1功能缺陷植株均表现出极弱的长势，光合同化速率大幅度降低，PSⅠ和PSⅡ受到严重的光抑制，且对PSⅠ影响更明显，叶片CEF维持在极低水平，表现出几乎完全抑制Y(ND)，而在强光下PSⅡ仍能保持较高的NPQ，表明PGR5/PGRL1依赖的环式电子传递途径具有重要的光保护作用，且对PSⅡ和PSⅠ的光保护存在差异。②低夜温处理后，WT的Pn、E和GH2O显著的低于PGR5-OE和PGRL1-OE，叶绿体透射电镜观察结果表明PGR5/PGRL1依赖的CEF能够缓解低夜温对叶绿体质量造成的损伤。③PGR5-OE、PGRL1-OE显著提高了低夜温下番茄叶片PSI和PSⅡ活性，以及ETR(I)、ETR(II)和CEF，通过增强Y(ND)和Y(NPQ)缓解了低夜温光抑制程度；表明PGR5/PGRL1依赖的CEF能够通过加强光保护作用缓解低夜温对番茄叶片光合性能的抑制。