多效性QTLs Ghd8和Ghd7.1调控的水稻穗发育和株型建成的基因网络解析

Rice grain yield is a key factor affecting national food security. Recently we have cloned the pleiotropic QTLs Ghd7.1 and Ghd8 with effects on spikelets per panicle, plant height and heading date, and found their genetic interaction effects on plant architecture. In this project, we will use the near isogenic lines of Ghd7.1 and Ghd8, their transgenic plants, mutants and the plants pyramiding both genes to elucidate the molecular mechanisms underlying panicle development and plant architecture. We will clarify their molecular basis of their interaction at the levels of transcription and proteins. We will construct yeast two-hybrid libraries each with young panicle, stem, leaf cushion and tillering buds as samples. We will isolate the proteins interacted with Ghd7 and Ghd8 by using the biological techniques of yeast 2-hybrid, chromatin immunoprecipitation followed by sequencing, DNase I hypersensitive site sequencing. We will explore the relationship between phytohormone and functional action of Ghd7 and Ghd8 by comparison of changing in pytohormone synthesis pathway or signal transduction pathway. Finally we expect to establish the gene regulatory network for panicle development and plant architecture. In addition, we will deep sequence Ghd7.1 and Ghd8 for a germplasm collection of 350 accessions, and analyze the genetic effects and adaptation of two-gene combinations. We will eventually establish gene regulatory networks for panicle development and plant architecture, and provide theoretical instructions for breeding high yield rice varieties.

水稻产量维系国家粮食安全。我们新近克隆了同时影响每穗粒数，株高和开花期的多效性QTLs Ghd7.1和 Ghd8，发现它们在遗传学上的互作显著改善株型。本研究，我们将以Ghd7.1和 Ghd8的近等基因系，转基因单株，突变体和二基因聚合单株为材料，从转录调控，蛋白互作等层面解析它们的互作机理。以幼穗，茎秆，叶枕和分蘖芽为样品，构建酵母双杂交筛选文库，通过酵母双杂交，染色质免疫共沉淀结合测序技术，以及DNase I 超敏感位点测序等分子生物学手段，分离与Ghd8及Ghd7.1互作的蛋白质；通过比较分析细胞分裂素，生长素等植物激素的合成途径和信号传导途径的变化，探索激素在Ghd7.1及Ghd8功能发挥过程中的作用。对350份资源种质材料进行Ghd7.1和Ghd8深度测序，在二位点水平上分析不同基因组合的效应和生态区分布特点。最终建立穗发育和株型建成的遗传调控网络，为水稻高产育种提供理论支撑。

我们对水稻穗发育和株型的分子调控网络更知之甚少。鉴于穗大小和株高性状的重要性，我们有必要开展穗发育和株型建成分子调控机制的研究。Ghd7.1和Ghd8作为同时调控株型和穗发育的关键因子，剖析它们的分子调控网络不仅让我们理解这两个性状的分子机理，而且为水稻高产育种提供理论支撑。.本研究通过酵母双杂交文库筛选，发现所有与Ghd8互作的蛋白都是HAP蛋白家族，进一步分析发现，Ghd8可以与9个HAP2和HAP5家族成员互作，推测Ghd8在水稻中主要是作为HAP3成员通过与HAP2和HAP5形成三聚体的复合物发挥功能。克隆了与Ghd7和Ghd8的互作基因Hd1，三基因组合显著延迟开花，大大限制了其合适种植区。通过对一批遗传变异丰富的材料开展三基因测序，发现不同基因型都非常适合于材料原产区，在原产区有较高的产量。根据不同生态区的光温条件推荐各自地区高产稳产的基因型组合。利用酵母双杂交和BiFC方法证明了Ghd7与Hd1存在蛋白水平的互作，最终确定Hd1(32-111)和Hd1(112-337)与Ghd7(187-257)存在显著互作。也即互作是通过Ghd7的CCT结构域与Hd1的锌指结构域和112-337氨基酸区域结合来实现的。多种生物技术手段证明Hd1具有转录激活活性，而Hd1的转录激活活性受Ghd7抑制。..Ghd7、Ghd7.1和Ghd8主要是通过延长生育期，使赤霉素合成途径基因工作时间延长，导致株高增加。它们不改变穗发育历程，但是会增大幼穗原基的大小，激活幼穗枝梗原基特别是二次枝梗原基的活性，从而增加每穗粒数。多种激素喷施试验表明，细胞分裂素与Ghd7.1和Ghd8存在调控关系。结合SELEX和RNA-seq技术筛选到Ghd7.1的下游直接候选靶标。另外，我们分析了FZP调控穗发育的机制，发现FZP参与调控class B类基因、AGL6类基因以及class E类基因的表达，进而实现对水稻花器官的调控。同时利用全基因组关联分析定位了水稻穗形相关的数量基因30个，克隆了分蘖角度基因TAC3，补充了基于本研究有限材料的穗形态和株型建成理论。.总之，通过3年的研究，我们初步明确了Ghd7、Ghd8和Ghd7.1基因控制水稻穗发育和株型建成的分子调控网络，为理想株型和高产育种提供理论指导。