大穗基因LP调控水稻穗型发育的深度解析及应用研究

Rice is one of the three major staple food crops in the world. It is critical to maintain and increase the rice grain yield for ensuring China's food security and sustainable development of the national economy. In our previous research, we have identified two allelic larger panicle (lp-1, lp-2) mutants, which show a typical phenotype in panicles such as larger panicle, more grains per panicle and the improved grain yield. Moreover, the plant architecture of mutants has also greatly improved, with the characteristics of the ideal plant type, suggesting that mutant plants have useful potential for high grain yield in rice breeding. Map-based cloning reveals that LARGER PANICLE (LP) encodes a Kelch repeat-containing F-box protein, which is a key component in the SCF complex, functions as a receptor to recruit specific phosphorylated substrates for ubiquitylation and degradation. By using EMS mutagenesis, we also obtained the reverse mutant (also named as second site mutation) of lp-1, named as slp. However, at present, neither the specific substrates recruited by LP nor the LP-interacting protein has been found yet. In this proposal, we plan to analyze the function of LP in modulating the rice panicle development in depth. In order to screen the interacting proteins of LP and its regulatory genes, methods such as the yeast two-hybrid, protein two-dimensional electrophoresis coupled to mass spectrometry sequencing, map-based cloning are all involved in this proposal. At the same time, the potential value of mutant plants for rice high yield breeding will also be verified by hybridization among elite varieties and backcrossing. We believe that this study has theoretical and practical significance for cultivating new high-yielding rice varieties.

水稻是三大粮食作物之一，提高水稻产量对保证我国粮食安全和国民经济可持续发展具有十分重要的意义。在前期研究中，我们获得了两个等位的大穗突变体，其典型特征是穗大、粒多、产量提高。此外，植株株型也有很大的改良，具有理想株型的特征，预示着突变体有良好的应用前景。通过图位克隆，我们已经获得了大穗基因LP, 其编码一个F-box 蛋白，在SCF 复合体中起识别底物蛋白的作用，可能参与蛋白的泛素化降解途径。利用EMS诱变，我们还获得了大穗突变体的回复突变体slp。目前LP作用底物尚不明确，其在水稻穗型发育中的分子机理尚不清楚。本研究拟在前期研究基础上，利用酵母双杂、蛋白双向电泳偶联质谱测序、回复突变基因克隆等研究手段，筛选LP蛋白的互作蛋白及其调控基因，并通过品种间杂交和回交，开展LP基因的育种价值研究。通过上述研究，以期深入解析LP蛋白调控穗型发育的分子网络，为水稻优质高产新品种的培育提供理论指导。

水稻是世界上最重要的农作物之一，提高水稻产量对保证我国粮食安全和国民经济可持续发展具有十分重要的意义。在前期研究中，我们获得了两个等位的大穗突变体，并通过图位克隆获得了大穗基因LARGER PANICLE (LP)，编码一个F-box蛋白，在SCF复合体中起识别底物蛋白的作用。本研究在此基础上，利用酵母双杂、蛋白双向电泳偶联质谱测序筛选获得两个LP的互作蛋白OSK1和INACT1，OSK1被注释为SKP1-like protein 1B，是水稻SCF复合体组分之一；而INACT1是一个动植物中保守的转录因子。酵母双杂交及荧光双分子互补法验证发现OSK1与LP的F-box结构域互作，而INACT1很可能与LP的C端结构域互作，是LP蛋白正真的作用底物。CRISPR-Cas9实验证明OSK1的突变会使水稻植株致死，表明OSK1在水稻整个生长发育阶段的重要作用。其次，我们通过图位克隆鉴定获得LP抑制因子Suppressor of LP(SLP)。SLP基因编码一个具有APETALA2 DNA结合结构域的植物特异转录因子，是BR信号转导途径中的正向调控因子。lp-1回复突变体(lp/lp slp/slp)中，SLP突变后不再响应BR信号，植株产生稻穗变小，单株穗粒数减少的表型。再次，通过品种间杂交和回交，将优异等位基因lp-2作为供体亲本导入盐稻8号，发现能提高受体的单株产量。. 通过上述研究，我们不仅筛选到LP蛋白的互作蛋白OSK1及可能底物INACT1；还获得了LP的抑制因子SLP。较完善地解析了LP蛋白调控穗型发育的分子网络，为水稻优质高产新品种的培育提供理论指导。同时通过杂交和回交实验验证lp-2具有良好地应用前景。. 在原有项目计划基础上增加了部分研究工作：SRL2 (Semi-Rolled Leaf2)基因的克隆及功能研究。SRL2基因编码一个新的植物特异的功能未知蛋白，主要在叶片维管束区域表达。我们的实验结果证明SRL2基因很可能通过调控YABBY类基因的表达来调控叶片厚壁细胞的分布及分化，进而影响水稻植株株型及穗型。