响应调节因子SlRR17介导茉莉酸信号调控番茄花粉发育的分子机制研究

In angiosperm plants，male gametophyte development is one of the most import developmental events. Tomato，one of the most important vegetable crops grown around the world, is regarded as an ideal model plant for reproductive development study. The pollen development process is highly sensitive to environmental stress in tomato. Under protected culture, the male sterility induced by adverse temperature conditions had a great influence on tomato yield and quality. However, the signal transduction mechanism of tomato pollen development process is not clear. In previous study, we have identified all the two-component system (TCS) genes in tomato genome, one of the main signal transduction pathway in plant, and then confirmed that an tomato species-specific response regulator SlRR17 can participate in the regulation of the pollen development in tomato. Based on these findings, in the present project, the function of SlRR17 and other B-type SlRRs in the same sub-family on tomato pollen development will be investigated through cell biology, genetics and physiological and biochemical analysis. The target genes regulated by SlRR17 were screened by RNA-seq analysis, ChIP-qPCR and protein hybridization. The effects of SlRR17 on TCS signaling pathway were investigated by yeast two-hybrid and phosphorylation experiments. The interaction of TCS and jasmonic acid (JA) signal mediated by SlRR17 was studied by using hormone treatment and biochemical analysis，in addition, the role of the interaction between TCS and JA was clarified on the high temperature-induced pollen abortion. The results will provide a basis for further understanding the molecular regulation mechanism of pollen development in tomato, and even the molecular mechanism of male sterility induced by high temperature stress. Finally, it will provide a new strategy for effective regulation of crop fertility and improving fruit set stability, which is of great significance to tomato breeding and culture.

番茄花粉发育对环境胁迫极度敏感，在设施生产中逆温诱导的雄性不育对番茄产量与品质影响很大，但番茄花粉发育过程的信号传递机制尚不清楚。本项目拟在分离番茄双组分信号系统基因，并初步证实响应调节因子SlRR17可参与调控番茄花粉发育的基础上，利用细胞生物学、遗传学及生理生化技术明确SlRR17与同亚族B型SlRRs在番茄花粉发育中的功能；通过酵母双杂以及磷酸化实验探索SlRR17在TCS调控花粉发育的信号通路；利用RNA-seq、ChIP-qPCR以及蛋白杂交等技术筛选SlRR17作用的靶基因，并进行功能验证，以阐明SlRR17参与JA信号调控花粉发育与高温胁迫响应的分子机制，明确SlRRs在TCS与JA信号交互应答中的作用。研究结果将为深入阐明番茄花粉发育的基因调控网络以及高温下雄性不育发生的分子机制提供研究基础，最终为有效调控作物育性，提高番茄坐果稳定性提供新途径，对作物抗性育种具有重要意义。

番茄是全世界栽培最为普遍的蔬菜之一，在我国蔬菜生产与消费中占有重要地位。作物花粉发育过程对环境胁迫具有高度敏感性，胁迫诱导的花粉败育对于番茄产量与品质有极大影响。但作物花粉发育以及对外界环境响应的信号传递机制并不清楚。目前有关细胞分裂素在植物花药与花粉发育中的作用目前还鲜有报道。本项目以番茄为材料，探索细胞分裂素响应调节因子SlRRs在花粉发育以逆境响应过程的作用以及调控通路。通过对31个SlRRs家族基因时空表达以及对胁迫响应特征分析，鉴定了TCS 新组分SlRR17（SlRR13）等花药中优势表达的基因；利用CRISPR/Cas9以及过表达技术研究了SlRRs家族基因在番茄生长与花粉发育过程的生物学功能，证实了SlRR17（SlRR13）参与低温下花粉发育过程，其功能的缺失导致植株对低温胁迫更敏感，说明细胞分裂素信号传导途径对低温下花粉发育十分重要；转录组分析与外源激素处理实验证实其可能通过调控赤霉素合成与茉莉酸信号传导基因来参与低温下花粉发育过程，提出了细胞分裂素信号组分整合多种激素信号调控花粉发育的分子模型。利用单基因敲除与多基因敲除突变体材料，通过遗传分析证实了SlRR6/SlRR7（ARR17同源基因）在番茄花粉发育过程中存在功能冗余，共敲除SlRR6/SlRR7基因导致花粉败育，但是SlRR6或SlRR7单独敲除并不影响番茄雄性发育。SlRR6单敲导致植株矮化、叶片变小，证实其在番茄生长与发育过程具有多重功能，蛋白杂交证实SlRR6和SlSAUR58之间存在互作关系，而SlRR6 通过影响 GA 和 IAA 稳态影响细胞扩增和细胞分裂正向控制植物生长；通过对下游靶基因的功能分析，建立了调控分子模型；项目最终还提出了创建番茄可调控核雄性不育系的新方法与利用途径，为降低番茄制种成本提供技术支撑。