拟南芥B3家族转录因子ABS2调控花瓣发育的机理研究

Flower is the reproductive organ of angiosperms. As the most prominent part of the flower, petals are of great economic and cultural importance. In our previous work, we isolated an Arabidopsis B3 transcriptional factor gene ABS2. Over-expression of ABS2 led to a loss-of-petal phenotype, while triple loss-of-function mutants of ABS2 and its two closest homologous genes produced flowers with increased numbers of petals. In addition, ABS2 over-expression mutants showed abnormal auxin distributions in leaves as indicated by the expression of auxin reporter gene DR5-GUS. A “cup-shaped cotyledon” phenotype resembling the cuc1cuc2 double mutant was observed in strong ABS2 over-expression lines. These results suggest that ABS2 is involved in the regulation of petal development and auxin homeostasis. In this project, we propose to carry out the following experiments to further elucidate the functions of ABS2 in petal development. We will perform detailed analysis of petal phenotypes of both gain-of-function and loss-of-function mutants of ABS2 and its two homologous genes, and determine the expression patterns of ABS2 during early flower development. Furthermore, we will analyze auxin responses of ABS2 and determine the auxin localization and transport processes in the inflorescence meristem of ABS2 mutants. Finally, we will assess the genetic relationship between ABS2 and other flower and petal development related genes and determine the direct target genes of ABS2 using DEX/Cycloheximide (CHX) treatment and Chromatin Immuno-Precipitation (ChIP) experiments. Our results will not only gain new insight into the molecular mechanisms of petal development, but also provide new avenues for artificially engineering plant petal numbers in the future.

花是被子植物特有的生殖器官。花瓣是花的重要组成部分，具有重要的经济和文化价值。我们在前期研究中发现过表达拟南芥B3家族转录因子基因ABS2能使转基因拟南芥表现出花瓣缺失表型，而ABS2及其两个同源基因的三重功能缺失突变体出现花瓣数量增加表型。ABS2的强过表达系中出现类似cuc1cuc2双突变体的“杯状子叶”表型，并影响叶片中生长素的分布。这些结果暗示ABS2调控植物花瓣的发育并参与生长素稳态调控。本项目拟通过ABS2及其同源基因突变体的花瓣发育表型、ABS2基因在花瓣发育早期的精细表达模式分析、ABS2表达量变化对生长素在花分生组织中分布和运输的影响、ABS2与花器官和花瓣发育相关基因的遗传互作、ABS2相互作用蛋白的筛选以及ABS2靶点基因的筛选鉴定等研究，揭示ABS2调控植物花瓣发育的分子机制。本项目为阐明植物花器官发育的分子调控机理提供理论贡献，同时为人工调控花瓣数量提供新的途径。

叶是植物进行光合作用的主要器官，对于植物和人类都具有重要意义。花是叶的变态，我们前期克隆了一个拟南芥B3家族RAV亚家族转录因子基因ABS2/NGAL1，ABS2的过量表达导致无花瓣的表型，本项目在前期研究的基础上，对NGAL1及其同源NGAL2和NGAL3的功能进行了深入研究。通过本项目的研究，发现分别独立过表达NGAL1-3，在营养生长阶段，能够导致杯状子叶的表型（cup-shaped cotyledon）。在生殖生长阶段，ngaltri三重功能缺失导致雌蕊发育异常表型，并能增强cuc2-1D花发育异常的表型。这些表型类似于cuc1cuc2双突变体的表型，CUC基因是目前已知植物叶片边缘发育的关键调控基因。进一步研究发现，NGAL1-3过表达突变体具有叶片边缘平滑的表型，而NGAL1-3功能缺失三突变体ngaltri则表现为叶片边缘锯齿加剧的表型，这些结果暗示NGALs和CUC基因共同调控植物的生长发育。转录组测序比较分析结果表明包括CUC家族基因在内的许多基因的转录水平受到NGALs的调控。遗传学研究发现，CUC2作用于NGALs下游，且NGALs的转录调控作用依赖于CUC2。遗传研究和RNA-seq分析从遗传学和分子水平发现，NGALs调控叶边缘的发育依赖于CUC2，且NGALs对CUC2基因的转录水平与miR164s途径无关。最后利用拟南芥原生质体的报告基因系统检测发现NGAL1能够直接结合CUC2的启动子，并抑制CUC2基因的转录。通过这些工作，进一步加深了对B3家族RAV亚家族的三个转录因子NGAL1-3在植物生长发育中功能的认识，提出了一个NGAL1通过转录抑制负调控CUC2的分子作用模型。