水稻微效QTL qGL1.2调控粒长和粒重的分子机理研究

Grain appearance and grain weight are controlled by naturally occurring quantitative trait loci (QTLs). While considerable efforts have been made to fine-map and clone major QTLs for grain appearance and grain weight, it is not until recently that the attention has been paid to the minor QTLs. A growing number of experimental evidences indicated that minor QTL also play important role in trait regulation, and the increase in rice yield over the past three decades illustrated that minor QTL for grain weight contributes to rice variety improvement. Previously, a minor QTL, qGL1.2, was delimited to a 40.9 kb region on the long arm of chromosome 1, using near isogenic lines (NILs) derived from Zhenshan973/Milyang46. The genomic sequences of two ORFs contained in the target region were sequenced. As compared with Zhenshan97 homozygous lines, grain length and 1000-grain weight in Milyang46 homozygous lines increased by approximately 0.1 mm and 0.5 g, respectively. This proposal intends to isolate qGL1.2 by map-based cloning. Firstly, Complementation test will be conducted and knock-out transgenic rice plants will also be generated to confirm the function of the candidate gene. Secondly, subcellular localization and analysis of promoter activity will be conducted to investigate the expression pattern of qGL1.2. Thirdly, The spikelet hulls of NIL set will be observed using scanning electron microscope to detect the effect of qGL1.2 on cell development of spikelet hulls. Fourthly, RNA-seq will be employed to analyze the signaling pathway of qGL1.2. Fifthly, haplotype of qGL1.2 in rice germplasm will be analysis. Finally, effect of qGL1.2 for other grain quality traits and yield traits will be investigated to evaluate its breeding potential. Results of our research are expected to provide new knowledge for regulation network of grain weight in rice and facilitate the utilization of these minor QTL for breeding practice.

水稻粒形和粒重一直是QTL研究的重要对象，前人工作多围绕主效座位开展。越来越多证据显示微效QTL在目标性状分子调控中亦扮演重要角色；水稻近三十年增产历程也暗示微效QTL对品种改良具有重要作用；但尚未有粒形/粒重微效QTL克隆。前期应用珍汕97/密阳46衍生的近等基因系，将微效QTL qGL1.2界定在包含2个ORF的40.9 kb内，并完成了候选基因测序；该座位主要控制粒长和粒重，相比母本型株系，父本型粒长增加0.08~0.14mm，千粒重提高0.34~0.60g。本项目拟开展qGL1.2图位克隆：构建转基因植株，验证候选基因功能；进行基因表达模式分析、颖壳细胞电镜观察、转录组测序，探索它调控粒形/粒重的分子机理；测序稻种资源，分析它的等位基因变异及其进化关系；鉴定它对品质和产量性状的效应，评估该基因的育种价值。本研究旨在进一步完善对水稻粒形/粒重遗传基础的认识，并为水稻品种改良提供参考。

前期应用珍汕97/密阳46衍生的近等基因系，精细定位一个控制水稻粒长和粒重的微效QTL qGL1.2。在本项目中，我们针对该QTL的候选基因，应用CRISPR/Cas9系统创制了基因敲除突变体，并构建了遗传互补转基因植株，确认OsVQ4为qGL1.2的目的基因。与近等基因系两种纯合基因型株系间的差异相比，基因敲除材料与野生型间的粒长差异增加了2.7~3.5倍，粒重差异增加了5.0~6.9倍。这说明，应用基因编辑手段可以在微效QTL上创制出主效等位基因。亚细胞定位发现，qGL1.2蛋白主要于细胞核中表达。组织表达分析发现，qGL1.2在叶片中表达最高，其次为幼穗。颖壳扫描电镜观察发现，qGL1.2可能通过影响颖壳细胞发育影响粒形。转录组和基因表达分析显示，qGL1.2可能作为转录因子调控蛋白，通过调控OsLG3等基因的表达影响粒长和粒重。品种资源分析发现，qGL1.2主要存在3种单倍型。农艺性状考查发现，qGL1.2在控制粒重的同时，对每穗粒数、抽穗期和整精米率具有显著影响；由于对粒重和粒数的效应方向相反，该QTL对单株产量无显著作用。也就是说，qGL1.2可在不影响产量的条件下改良生育期，具有育种应用价值。这些结果显示，微效QTL克隆不仅有利于水稻农艺性状遗传基础的完整解析，而且可为水稻育种提供新基因资源。