水稻URL1基因调控叶片卷曲的分子机制研究

Leaf is the major organ for photosynthesis in rice plant. The improvement of leaf morphology is one of the main targets of rice breeding for ideal plant architecture. Moderate leaf rolling is beneficial for the improvement of photosynthesis rate and grain yield. Although many genes controlling leaf rolling have been cloned in rice, the molecular mechanism underlying leaf rolling is still limited to individual genes and fragmented. More regulatory genes are needed to be cloned to construct detailed and refined molecular regulation network of leaf rolling. Furthermore，most of the cloned genes controlling leaf rolling have not been effectively used in rice breeding, due to some undesired effects on yield parameters. In this project, the semi-dominant mutant upward rolled leaves (url1) was selected from a collection of rice mutants with variant leaf morphologies. The url1 mutant exhibits erect and rolled leaves without side effects on panicle morphology, fertility and grain yield. We cloned the responsible gene URL1 using a map-based cloning approach which encodes a transcription factor. The expression level of URL1 was significantly increased in the url1 mutant. The URL1 gene was confirmed to control rice leaf rolling via downregulation of URL1 expression in the url1 mutant and overexpression in the wild type. We have identified two downstream genes directly regulated by URL1 and two proteins interacting with URL1. We will use genetic, biochemical and molecular technology to reveal the molecular mechanism of URL1 and its interacting proteins in controlling leaf rolling. We will also evaluate the application potential of the URL1 gene and the url1 mutant in the rice high-yield breeding.

叶片作为水稻光合作用的主要场所，其形态改良是水稻株型育种的重要目标之一。叶片适度卷曲有利于提高光合效率和产量。目前虽已在水稻中克隆获得多个卷叶基因，但局限于单个基因的功能研究，有待于克隆更多卷叶调控基因，构建更加完善的卷叶分子调控网络。另外，已克隆的多数卷叶基因突变后伴随一些不利产量性状，因此没能在育种中得以有效利用。本项目选取了一份水稻半显性内卷叶突变体url1，其株型直立且穗型和育性等产量指标未受影响。目前图位克隆获得了候选基因URL1，该基因编码一转录因子，在突变体中表达量升高。通过在突变体中减量表达和野生型中超表达URL1，验证了URL1调控叶片卷曲的功能。初步筛选到两个URL1的互作蛋白和两个受URL1直接调控的下游基因。将通过遗传、生化及分子生物学的方法研究URL1及其互作蛋白调控叶形的分子机制，构建和完善叶形调控网络，评价URL1基因及其突变体材料在水稻高产育种中的应用价值。

叶片作为水稻光合作用的主要场所，其形态改良是水稻株型育种的重要目标之一。叶片适度卷曲有利于塑造紧凑株型、改善群体结构、提高光合效率和产量。目前虽已在水稻中克隆获得多个控制叶片卷曲的基因，但是各个基因在遗传和生化水平的关系仍然未知，有待于克隆更多基因，构建和完善水稻叶片卷曲分子调控网络。另外，已克隆的多数卷叶基因突变后伴随矮化、育性降低等不利产量性状，因此没能在育种中得以有效利用。. 本研究利用 EMS 诱变粳稻品种日本晴，获得了一份半显性内卷叶突变体 Url1 (Upward rolled leaf1)，其叶片上表皮泡状细胞数目和面积减少，导致叶片内卷，因而该突变体株型直立，但株高、穗型、育性及籽粒大小均未受到不利影响，适宜在高密度条件下种植。利用图位克隆的方法分离获得了URL1基因，该基因编码HD-ZIP IV家族转录因子ROC8。在Url1突变体中，该基因3’-UTR区特异且保守的CNS2 motif发生了一个单碱基替换 (C-T)，增强了其mRNA的稳定性，使得URL1基因在突变体中的表达量显著高于野生型。进一步生化分析表明，URL1可与另一HD-ZIP IV家族成员ROC5及转录抑制辅助因子TPL2形成转录抑制复合物，直接结合到泡状细胞发育正调控因子ACL1基因启动子上并抑制其表达。在Url1突变体中，采用基因编辑技术分别敲除TPL2或ROC5基因或超表达ACL1基因均可部分抑制其卷叶表型。这些结果表明URL1在水稻叶形调控中具有重要作用，并且其3’-UTR区特异且保守的CNS motif对HD-ZIP IV家族成员mRNA稳定性具有潜在的精细调控作用。该项研究有助于构建和完善水稻叶片卷曲分子调控网络，对水稻株型育种具有重要的理论和指导意义。