水稻穗型是最重要的产量性状之一。水稻穗型发育包括花序分生组织发生、一次和二次枝梗原基发生发育、小穗和颖花器官发生发育等过程，其中一次二次枝梗和小穗发育状态决定了穗大小和每穗颖花数。目前已克隆了至少28个水稻穗型或小穗发育相关的基因，但其调控和互作关系还不很清楚。我们从水稻获得了一个大穗型突变体lp2，其一次和二次枝梗数增多，每穗颖花数大幅增加（176.5%）。利用LP2lp2分离株系的分群混合高通量测序法，我们快速定位和克隆了目标基因为一个bHLH转录因子基因。本项目将研究该基因的生物学功能和遗传变异，分析对已知和未知的水稻穗建成发育相关基因的互作及其表达调控，揭示穗型发育的分子遗传调控网络。另一方面，基于其隐性遗传突变的特性，利用CRISPR/Cas9基因修饰系统对多个籼稻和粳稻优良品种的该基因进行功能弱化和功能缺失的修饰，培育更高产的新品种（系），探索新的高效作物分子育种技术方法。

Panicle architecture is one of the important traits for yield of rice. The panicle development in rice involves in the process of transition of stem apical meristem into inflorescence meristem and occurrences of primary and secondary branch meristems, spikelet meristem, and floral meristem. The developmental processes of primary and secondary branches and spikelet determine the grain number per panicle. So far at least 28 genes involved in panicle architecture development have been cloned, but the relationships of genetic regulatory and molecular interaction of the genes are largely unclear. We have identified a rice mutant of larger panicle, large panicle 2 (lp2) with significantly increased numbers of primary and secondary branches, and grains. By a strategy of high-throughput sequencing of bulked segregants of the M2 line for this mutation, we rapidly mapped and isolated the target gene that encodes a bHLH transcription factor. In this study, we will conduct these studies: (1) analysis of the molecular functions of this gene by transformation of constructs of over-expression and RNA interference; (2) analysis of the expression patterns of LP2 and its downstream genes related to panicle development; (3) screening the target genes and interactors of LP2; (4) analysis of the variation of cytokinin in young panicles of lp2 mutant and wildtype rice; (5) revealing the genetic regulatory network in controlling the panicle development; (6) examination of the allelic variation of LP2 in rice varieties with different panicle size (grain number per panicle) and the association between the allelic variation and panicle size. In addition, based on the recessive genetic feature of lp2, we will use CRISPR/Cas9 system to modify this gene in elite rice varieties to create weak allele or loss-of-function mutation, in order to develop a new and high-efficient technology of molecular breeding in crops.

株型、株高和穗型发育是作物高产育种的重要农艺性状。本项目利用一个水稻穗型突变体进行基因定位，在定位区选择17个候选基因进行基于CRISPR/Cas9基因编辑技术的功能敲除。其中一个编码锌指结构蛋白的基因OsZFN421的敲除产生了株型和穗型等多种性状发生了变异，包括株高降低了24.4%，穗长减少了33.3%，穗粒数减少了36.7%，粒重减少26.0%，结实率有所下降。该突变系的伸长茎的细胞长度与野生型的差异不显著，其茎的缩短主要是细胞数目减少。OsZFN421定位到细胞核。将oszfn421突变体和野生型日本晴的小苗进行了转录组测序，分析发现赤霉素GA相关的双萜合成途径的基因表达量差异最明显，表明oszfn421突变性状与GA合成途径可能密切相关。分析了oszfn421突变体与各种激素响应的关系，发现与油菜素内脂无关；发现突变体在喷施了GA3后株高高于清水对照，说明突变体对外源GA3有响应，植株内GA受体活性正常。而突变型株高接近多效唑（GA抑制剂）处理的野生型株高，推测矮化表型可能是内源赤霉素合成减少导致的。进一步测定突变体小苗具有生物活性的内源赤霉素GA1及GA3含量和其它种类GA含量，发现突变体的GA1及GA3含量和其它GA都较野生型含量低。并且,GA合成途径的第一个产物GA12及其下游各种GA产物在突变体的含量均比野生型的含量低，暗示oszfn421突变造成矮化成因是抑制了催化GA12合成的上游因子。本项目克隆的转录因子基因OsZFN421在调控水稻细胞发育，进而在控制植株的株型株高，穗型，粒型等多种性状中发挥重要作用。该基因可能通过调控GA代谢的遗传网络发挥作用，因此是一个重要的GA代谢调控子。通过对该基因进行表达调控，可望在水稻遗传改良中具有重要的应用价值。