光合电子受体铁氧还蛋白调控水稻抽穗期的分子机理研究

Ferredoxin (Fds) protein as unique electron acceptor, involved in variety of fundamental metabolic and signaling processes, which is indispensable for plant growth. The molecular mechanisms of Fd such as regulation of electron partitioning, impact of photosynthetic rate and involvement in the carbon fixing remain elusive in rice. Here we reported a heading date delay and yellowish leaf 1 (hdy1) mutant derived from Japonica rice cultivar 'Nipponbare' subjected to EMS treatment. In the paddy field, the hdy1 mutant appeared significantly late heading date and yellow-green leaves during the whole growth stage. Further investigation indicated that the abnormal phenotype of hdy1 was connected with depressed pigment content and photosynthetic rate. Genetic analysis results showed that the hdy1 mutant phenotype was caused by a single recessive nuclear gene mutation. Map-based cloning revealed that OsHDY1, located on chromosome 3, and encodes an ortholog of the AtFdC2 gene. Complementation and overexpression, transgenic plants exhibited the mutant phenotype including head date, leaf color and the transcription levels of the FdC2 were completely rescued by transformation with OsHDY1. Real-time PCR revealed that the expression product of OsHDY1 was detected in almost all of organs except root, whereas highest expression levels were observed in seeding new leaves. The lower expression levels of HDY1 and content of iron were detected in hdy1 than WT’s. The FdC2::GFP was detected in the chloroplasts of rice. Real-time PCR results showed that the expression of many photosynthetic electron transfer related genes in hdy1 were higher than WT. These results suggest that OsFdC2 plays an important role in photosynthetic rate and development of heading date by regulating electron transfer and chlorophyll content in rice. In this project, base on the previous studies, we will further identify the regulatory mechanisms for FdC2 in photosynthetic electron transfer chain of rice using CRISPR-Cas9, yeast two - hybrid system, RNA-seq, BiFC and pull down. Transgenic experiments with different constructs of OsFdC2 and the morphology, genetics and physiology of hdy1 involved will be analyzed. The result will important to understand the mechanism of photosynthetic electron transfer chain of rice, to establish theoretical basis for tapping photosynthetic potential of C3 plant and for better yields of rice.

铁氧还蛋白（Fds）作为光合电子传递的关键电子受体，参与碳代谢、N同化、S同化等诸多生物学反应。但是Fds如何协调不同生物学反应中电子的传递和平衡？对植物表型产生何种的影响，人们还知之甚少。项目组前期通过图位克隆的方法，在水稻Chr.3克隆了一个抽穗期调控基因，基因突变后引起严重的抽穗延迟、叶片失绿等表型。互补和过表达实验已经验证了该基因的功能。基因结构分析表明该基因编码C末端延伸的铁氧还蛋白（FdC2），GFP融合蛋白定位于叶绿体，基因主要在叶片中表达。表达及其他分析结果显示OsFdC2可能是水稻主要的PSI电子传递体。本项目拟通过酵母双杂、BIFC、CHIP、CRISPR-Cas9、各种突变体及转基因材料的生理生化分析等实验，对OsFdC2功能进行深入研究，探索其在水稻光合电子传递过程中的分子调控机制，为挖掘C3植物光能利用潜力，提高水稻、小麦等主粮作物产量奠定理论基础。

铁氧还蛋白（Fds）作为光合电子传递的关键电子受体，参与碳代谢、N同化、S同化等诸多生物学反应。项目组前期克隆了一个具有C末端延伸的铁氧还蛋白（FdC2），该基因突变后会造成水稻严重的抽穗延迟、叶片失绿等表型。以此为基础本项目利用同源序列比对在水稻中找到了FdC2另外6个同源蛋白，其中包括5个Fd蛋白和一个有C末端延伸的FdC1蛋白。聚类分析结果表明只有Fd1可能为叶型Fd,而另外4个均被预测为根型Fd，由于C末端延伸序列，FdC1和FdC2单独成簇。不同Fds在叶片中的表达证明，与Fd1相比，除了FdC2表达量约为Fd1的15%外，其他Fds的表达都非常低，仅为Fd1表达量的0.1-2%，几乎可以忽略不计。结合fd1和fdc2突变体中各个Fds基因的表达，我们推测水稻中Fd1为主要的光合电子传递蛋白，FdC2可能在某种条件下提供补充。通过原核表达我们在体外用细胞色素c和NADP+光还原实验验证了Fd1和FdC2的光合电子传递能力，证明二者均可从PSI接受电子，Fd1可为碳固定提供还原力，而FdC2则不能。为了探索FdC2的电子受体，我们通过酵母文库筛选共获得53个阳性克隆，包括29个可能的互作基因，其中3次以上互作的基因有7个，与光系统I相关的基因有3个。为了进一步验证互作的正确性，我们首先对几个重点候选基因进行了亚细胞定位分析，结果表明4个候选基因有3个定位于叶绿体。随后我们又构建了GST和His标签载体、BIFC、LUC及定点突变载体，并分别进行了Pulldown等体内、体外验证，同时将定点突变载体转化水稻日本晴愈伤，构建互作基因的定点突变体。研究结果显示拟南芥循环电子传递关键基因PGRL1同源基因和PSI核心亚基与FdC2均有强烈的互作。其余互作基因的体内、体外验证及定点突变、过表达转基因株构建和鉴定试验正在进行中。后续我们将进一步明确Fd1和FdC2不同的电子受体，进而揭示其分子作用机理。本项目的运行为深入探索水稻不同Fds的功能提供了有益的线索，也为光合作用分子机理的揭示提供了理论支撑。