一个新的水稻穗顶端颖花退化基因的克隆和功能鉴定

The abortion of spikelet reduces the number of kernel and seriously affects yield, which is a very unfavorable trait for rice production. So far, there are few studies on the genetic and molecular mechanisms of the abortion of rice spikelet, and the molecular mechanism of spikelet abortion is not clear. It is of great importance to mapping and clone the genes for spikelet abortion, and elucidate the molecular regulating mechanism, then, provide gene and theoretical basis for genetic improvement of yield. In this proposal, we have obtained a rice mutant (spa7), which spikelets on panicle top were aborted. Genetic analysis showed that it was controlled by a dominant nuclear gene, gene mapping indicated that spa was located in 37.5kb region between marker S-8 and S-12 on chromosome 7, no gene for regulating the abortion of rice spikelet has been reported in this region before. A candidate gene was found through gene prediction, sequencing and expression analysis. Based on these findings, we would identify the candidate gene of spa7 through transgenic complementation test and CRISPR/Cass9 gene editing to create targeted knockout materials, analyze the function of spa7 during panicle and spikelet development through morphological anatomy, molecular biology, biochemistry and other genetic means, elucidate the molecular mechanism of spa7 gene to regulate the abortion of top panicle spikelet, so as to providing theoretical basis to avoid spikelet abortion in rice by molecular breeding.

颖花退化减少穗粒数，严重影响产量，是对水稻生产非常不利的性状。迄今关于水稻颖花退化的遗传和分子机理研究非常少，对其发生的分子调控机制不清楚。定位和克隆颖花退化基因，阐明其分子调控机制，为产量遗传改良提供基因和理论基础，具有重要意义。本研究前期获得了一个水稻穗顶端颖花退化突变体spa7，遗传分析表明受一对显性核基因控制，基因定位在第7染色体标记S-8和S-12间37.5kb区域，该区域未见有调控水稻颖花退化的基因报道。通过基因预测、测序和表达分析，获得了候选基因。本研究在此基础上，通过转基因功能互补试验和CRISPR/Cass9基因编辑创造靶向敲除材料，验证spa7候选基因；通过形态组织解剖学、分子生物学、生物化学和遗传学等手段，研究spd7基因在水稻幼穗颖花发育过程中的功能，解析其调控水稻穗顶端颖花退化的分子作用机制，为改善水稻颖花退化特性的分子育种提供理论依据。

颖花退化减少穗粒数，是对水稻生产非常不利的性状。定位和克隆颖花退化基因，阐明其分子调控机制，为产量遗传改良提供基因和理论基础，具有重要意义。本研究鉴定了一个新的水稻穗顶端颖花退化主效位点qPAA7（即spa7）,通过连锁分析和QTL-Seq分析，将其定位在第7染色体73.8kb区域内，该区域有9个基因被功能注释，根据基因注释、表达谱和DNA序列差异，预测LOC_Os07g41220、LOC_Os07g41250和LOC_Os07g41280为可能的候选基因。利用CRISPR-Cas9创造候选基因的靶向敲除材料，确认LOC_Os07g41280是qPAA7的目标基因，编码6-磷酸葡萄糖酸内酯酶2。qPAA7基因启动子存在7个光响应元件、4个激素响应元件以及4个非生物胁迫响应相关元件，表明qPAA7的表达可能受光因素、激素因素和非生物胁迫的调控。6-磷酸葡萄糖酸内酯酶2在磷酸戊糖氧化途径中起作用，可定位于质体和过氧化物体中，过氧化物酶体是细胞ROS产生的主要来源之一，突变体发育小穗中的过氧化氢含量较野生型极显著增加，表明过氧化氢含量积累导致了穗顶端小穗退化，但具体机制还需要进一步研究。形态组织解剖学分析表明qPAA7穗顶端退化发生在幼穗发育早期，细胞内发生裂解，导致细胞结构异常。在穗退化发生的三个关键时期取幼穗进行RNA测序，在野生型和突变体之间共鉴定到7677个DEGs，在三个时期均差异表达的基因有321个。GO和KEGG分析发现，碳水化合物、转录、酰胺生物合成的过程、核糖体、结构分子活性等13个生物学过程，昼夜节律、核糖体等2个KEGG通路在其中的两个时期同时富集，这些共同富集到的生物学途径可能参与了穗顶端颖花退化发生。