番茄PIL转录因子在高温诱导的番茄柱头外露中的作用及调控机理研究

Tomato，one of the most important vegetable crops，is widely grown in the world. Blossom drop is a common tomato growing problem. The most frequent cause of tomato blossom drop is sub-optimum temperature. In hot seasons and under protected culture, the stigma exsertion often occurs and causes the unsuccessful self-pollination, and finally markedly lowers fruit set. Therefore, it is important to study the molecular mechanisms regulating high-temperature response in tomato flower. In previous study, two phytochrome (phy)-interacting basic helix-loop-helix transcription factor (PIF)-like genes were identified by RNA-Seq analysis in tomato, named SlPIL3 and SlPIL4, which exhibited quite different expression pattern in tomato style and stamen under high temperature condition compared with normal condition. Based on these findings, the project will study expression profiles of S1PIL3 and S1PIL4 and some other related S1PILs using qRT-PCR and in situ hybridization and examine the subcellular location with transient expression assay. We will characterize their stress-responsive expression pattern under high temperature and some other abiotic stresses and try to establish the relationship between the transcription levels of S1PILs genes with the stigma-exsertion. Using amiRNA, chimera respressor and overexpression technique, we will elucidate the functions of S1PIL3 and S1PIL4 protein in tomato flower in response to high temperature. The diverse or redundant role of S1PIL3 and S1PIL4 in high temperature response in tomato is characterized. The downstream genes regulated by S1PIL3 or S1PIL4 i transcription factor are screened using RNA-Seq and bioinformation analysis using transgenic tomato plants. Meanwhile, the interacting proteins will be isolated through yeast two-hybrid and further proved using ChIP assay and DNA-binding analysis. Molecular interactions between PIF-mediated signal pathway and hormone signaling such as IAA and GA involving in plant growth and stress response is also analyzed based on hormone treatment and expression analysis. Finally, the molecular model of their regulation on stigma-exsertion under high temperature will be established. The results will provide the basis for exploring the morphological adaptaion mechanisms during reproductive stage under abiotic stress condition. The project also can provide the knowledge to develop the novel germplasm with heat-tolerance by genetic engineering in tomato and improve the culture methods under high temperature condition.

夏秋高温季节栽培番茄容易引起柱头外露导致自交授粉失败从而降低座果率，显著影响番茄产量。利用RNA-seq技术我们从高温胁迫的花柱与雄蕊中分离了两个差异表达的番茄PIF-like基因，SlPIL3与SlPIL4，为研究其在高温引发的番茄柱头外露过程的作用，利用qRT-PCR、原位杂交以及瞬时表达分析方法研究其组织表达特性与亚细胞定位; 利用CRES-T、amiRNA以及过表达技术研究其在番茄高温胁迫响应过程的生物学功能；利用高通量测序技术与蛋白杂交技术筛选其目标基因与互作蛋白，研究其在高温下转录调控机制；并通过激素处理实验探索SlPIL介导的信号与生长素等植物发育信号途径在高温胁迫响应过程的交互作用，建立SlPIL参与番茄高温诱导柱头外露的调控模式，以深入了解高温下植物生殖器官形态适应的控制机理，为利用遗传工程技术提高番茄在逆温下的坐果性能及抗高温栽培技术完善提供理论依据。

番茄是世界上普遍栽培的重要果菜类蔬菜，限制番茄坐果的主要原因是雄蕊与雌蕊发育和授粉时对外界环境极度敏感，尤其是对低温或高温的敏感性。高温易引起番茄柱头外露，由于番茄的异交授粉率不高于2%，花柱外露使得雌雄蕊无法正常授粉受精，造成座果率严重下降, 最终导致番茄减产，商品性下降。项目首先观察了高温对番茄雄蕊与雌蕊发育以及柱头外露的形态学、细胞学以及生理学影响，并评估了外源植物生长调节剂对柱头外露的调节作用，接着利用转录组结合组织切片、生化分析等方法鉴定了一批参与高温诱导番茄柱头外露调控的关键基因（其中包括PIFs转录因子）以及sRNAs, 提出了茉莉酸参与高温诱导番茄柱头外露的作用模型；在此基础上，研究了SlPIF3/4（原命名为SlPIL3/4）在生殖发育过程以及逆温胁迫过程的生物学功能，明确了SlPIF3与SlPIF4均可以调控番茄的生殖发育，但作用时期以及作用方式完全不同；SlPIF4参与高温下番茄雄蕊与雌蕊的生长过程。利用转录组测序RNA-seq分析与蛋白质杂交方法筛选SlPIF3与SlPIF4下游调控的重要基因以探索SlPIF3与SlPIF4在调控逆温下番茄生殖发育过程的作用机制，证实SlPIF4通过调控雄蕊发育基因的表达水平参与番茄逆温胁迫响应，而SlPIF3并不直接参与逆境胁迫响应过程。项目最终还建立了番茄SlPIFs参与调控逆温下番茄生殖发育的分子模型。