水稻主效基因Ghd7调控剑叶叶绿素含量的机制研究

Chlorophyll content in flag leaf is one of the most important physiological traits as it is closely related to leaf photosynthesis and crop yield potential. Grain number, plant height, and heading date7 (Ghd7) participates in multiple processes, including phase transition, architecture regulation, and stress response, and has been also demonstrated to be a major locus for natural variation of chlorophyll content at heading stage by our research group. Being a transcription factor, GHD7 has intrinsic transcriptional repression activity in vivo, however, the target genes and cis-acting elements remain unknown. Recently, by yeast one-hybrid assay, we have revealed that GHD7 can interact with the promoters of four genes encoding key enzymes of chlorophyll biosynthesis and light-harvesting chlorophyll a/b-binding proteins. In this research proposal, we plan to screen more candidate genes related to chlorophyll metabolism and light-harvesting, which were down-regulated by Ghd7 using qRT-PCR. The interaction of GHD7 protein with the promoters of candidate targets will be tested and verified through various approaches, such as yeast one-hybrid assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation. The key cis-regulatory elements of GHD7 will be determined by designing a series of truncated and site-mutated promoters. The expression profile of Ghd7 and its target genes and how the trait of chlorophyll content is affected will be elucidated, and the genetic interaction mode between Ghd7 and its targets will be characterized. The research results will help understand in depth how Ghd7 functions and provide guidance for rice breeding.

剑叶叶绿素含量与叶片光合作用及产量潜力密切相关，是水稻育种中非常重要的生理性状。Ghd7是控制水稻抽穗期、株高和穗粒数的主效QTL，参与了时期转换、株型调控及胁迫反应等多个过程，本研究小组还发现它是控制剑叶叶绿素含量自然变异的主效位点。Ghd7编码转录因子且具有转录抑制活性，但其直接调控的靶基因及顺式作用元件一直未知。本项目申请人利用酵母单杂技术首次鉴定出4个在启动子区与Ghd7存在直接互作编码叶绿素合成过程中的酶及叶绿素a/b结合蛋白的靶基因。本项目拟进一步通过qRT-PCR、酵母单杂、凝胶阻滞及染色质免疫共沉淀等技术鉴定验证与转录因子GHD7直接互作的叶绿素代谢或光能捕获相关的靶基因，采取启动子截短及定点突变的方法确定关键顺式作用元件，阐明Ghd7与靶基因的表达模式及与叶绿素含量等表型的关系，并研究Ghd7与靶基因的遗传互作。本研究对于深入理解Ghd7的功能及指导育种实践具有重要意义。

剑叶叶绿素含量与叶片光合作用及产量潜力密切相关，是水稻育种中非常重要的生理性状。本研究小组前期发现Ghd7是控制水稻剑叶叶绿素含量自然变异的主效位点，并鉴定出几个靶基因。本项目对Ghd7下调的关键靶基因OsL3 （编码叶绿素a/b结合蛋白）及OsFGH5（编码叶绿体基质蛋白）进行了功能研究，还分析了Ghd7介导的转录调控。.OsL3纯合突变体及RNAi材料叶绿素a、b及总叶绿素含量均极显著低于野生型。纯合突变体叶绿体发育严重受阻，类囊体片层结构稀疏且紊乱。通过酵母双杂交文库筛选，鉴定到1个调控叶绿素合成代谢的互作蛋白OsF。此外，还发现基因OGR在不同发育时期不同组织中表达模式与OsL3高度相似，其编码的蛋白也定位于叶绿体中。推测OsL3、OsF与OGR极可能形成了超级复合物以调控水稻叶绿素代谢。.与野生型相比，OsFGH5 CRISPR材料SPAD值、叶绿素总含量、叶绿素a及叶绿素b均出现极显著下降。OsFGH5突变体材料相较于野生型表现出更高的稳态NPQ值和较低的ETR水平。OsFGH5定位于叶绿体基质。利用酵母双杂交、BiFC、体外pull-down和体内Co-IP实验验证了水稻OsFGH5蛋白能与叶绿体ATP合酶CF1β亚基发生相互作用。.还利用RNA-seq研究了Ghd7超表达材料剑叶中的表达谱变化。对差异表达基因进行了GO富集分析。下调的差异表达基因主要与应激响应、蛋白修饰、激酶活性和定位质膜有关。上调的差异表达基因主要与光合作用、能量代谢过程、催化活性和定位类囊体相关。.此外，本研究还发现有功能的Ghd7等位基因增强了水稻的光保护能力，并改良了穗颈维管束系统。Ghd7与抽穗期基因Ehd1存在显著的遗传互作。.本研究对于深入理解Ghd7的功能具有重要科学意义，对于水稻育种实践具有重要指导意义。