一个新的水稻穗粒数关键基因的遗传发掘、图位克隆及功能分析

G46A, a heavy panicle CMS line, has more panicle branches and, as a result, showes dense panicle and more number of grain per panicle. To identify genes controlling number of grain per panicle in G46A, we isolated a sparse panicle mutant spp46 (sparse panicle 46) from G46B maintainer line through large-scale mutagenesis. The spp46 mutant exhibited reduced number of panicle branches and sparse density of grain, and its number of grain per panicle reduced 30% compared with wild-type parent G46B. Genetic analysis showed that the phenotype of the spp46 mutant was caused by a recessive mutation in a nuclear gene, which implied that the wild-type gene SPP46 would have greater effect on number of grain per panicle than Gn1a did, and functions as a positive regulator of grain number. Therefore, the SPP46 gene may be very useful in improving yield of rice. In the preliminary study, the spp46 mutant gene had been mapped on 72.6kb region of rice chromosome 3, and a candidate gene had been identified in this region through DNA sequencing, which revealed that it is a novel gene for number of grain per panicle. In this study, we will construct expression vector of the wild-type SPP46 gene, and confirm its function through transgenic technology. At the same time, we will further identify its biological function through investigation of mutant phenotypes in field, observation of panicle development, analysis of spatial and temporal expression patterns of SPP46, and subcellular localization of SPP46-GFP fusion protein. In addition, we will also investigate natural variation of the SPP46 locus in various types of rice varieties that have different grain density of panicles (such as dense panicle and sparse panicle) to identify its high yield allele. In a word, this study may provide gene resources with important value, and a novel and key control site in genetic network for yield traits of rice.

重穗型杂交稻骨干亲本不育系冈46A穗分枝较发达，表现穗大粒多、配合力高。为发掘冈46A的穗粒数基因，对冈46B保持系实施大规模诱变，成功获得稀穗突变体spp46。该突变体主要特征为穗分枝数显著减少，着粒密度明显变稀，每穗总粒数比野生型亲本减少30%（野生型比突变体则增加43%），遗传分析表明该突变性状受一对隐性核基因控制。前期研究已将该基因定位在第3染色体上72.6kb区域内，并通过测序发现了一个候选基因，且为新基因。本项目拟构建表达载体，通过转基因互补验证候选基因功能，同时，通过多年多点田间试验准确鉴定基因表型，通过幼穗发育过程观察、基因时空表达模式分析及表达蛋白亚细胞定位，探明该基因的表达特征和生物学功能，并进一步调查该基因位点在不同类型水稻品种中的自然变异情况，发掘其高产的等位基因，为水稻高产育种的分子设计提供重要价值基因资源，为解析水稻产量性状的遗传网络提供新的主要调控位点。

水稻产量主要由有效穗数、每穗粒数和粒重等三个因子构成，要提高水稻产量可以通过协调与适当增加穗数、每穗粒数和粒重等多条途径实现。重穗型杂交稻骨干亲本不育系冈46A穗分枝较发达，表现穗大粒多、配合力高。为发掘冈46A的穗粒数基因/主效QTL，对冈46B保持系实施大规模化学诱变，成功获得稀穗突变体spp46。该突变体主要特征为穗分枝数显著减少，着粒密度明显变稀，每穗总粒数比野生型亲本减少30%（换一个角度看，野生型亲本比突变体每穗粒数增加43%）。对spp46突变体与野生型亲本冈46B杂交F1、F2代及F2突变植株衍生的F3株系进行遗传分析，结果表明该突变性状受一对隐性核基因控制。利用分子标记及spp46突变体与着粒密度较大的粳稻品种中花11杂交、回交F2分离群体将该突变基因定位在第3染色体上72.6kb区域内，并通过基因注释和测序发现了一个候选基因，且为新基因，spp46突变体在该基因第3外显子区有1个碱基替换，导致了1个氨基酸的变化；再构建B4F2高代回交分离群体，将SPP46目标记基因精细定位在7.3kb区域内，该区域仅含有这个候选基因，从而进一步确认了该候选基因。构建含有SPP46启动子和基因组全长序列的转基因表达载体，通过农杆菌介导法导入高代回交突变株系，转基因后代表现穗粒数显著增加，同时，利用CRISPR/Cas9基因编辑技术敲除候选基因，获得转基因株系，结果显示该基因敲除后代表现出与spp46相似的稀穗突变表型，从而验证了该候选基因确为SPP46基因。另一方面，构建SPP46启动子+GUS报告基因(pSPP46::GUS)表达载体并导入水稻获得转基因株系，经GUS组织化学染色检测该基因表达模式，结果显示该基因在根、茎、叶片、叶鞘、幼穗、颖壳和颖果中均能看到明显的染色，表明它为组成型表达基因。此外，选取63份不同种质材料进行了SPP46基因组序列的自然变异调查，结果表明SPP46基因位点存在自然变异且可能与稻种进化有关。该基因位点可为水稻高产育种的分子设计提供重要价值基因资源，为解析水稻产量性状的遗传网络提供新的主要调控位点。