转录因子BolAHL16L在青花菜花球高产性状形成中的功能及分子调控机制研究

Curd is a specialized organ of broccoli (Brassica oleracea L var. italic), which is mainly resulted by overproliferation of inflorescence meristem. The formation of curd in floral development is the most remarkable trait that differentiates broccoli from other plant species. Moreover, curd is the important edible organ of broccoli. The quality and yield of curd is crucial for the economic value of broccoli. Utilization of heterosis (hybrid vigor) is the most important strategy to breed new broccoli varieties with high-yield curds. However, the molecular basis of curd development, especially the formation of the high-yield trait in curd remains largely unknown. In our previous study, a novel transcription factor from AHL（AT-hook motif nuclear-localized gene）gene family, named BolAHL16L, was demonstrated to be involved in high-yield heterosis of broccoli curd. The function analysis by overexpression of BolAHL16L in Arabidopsis indicated that BolAHL16L overexpression can significantly influence the organ development. The BolAHL16L overexpression transgenic Arabidopsis lines showed more flowering branches and flower buds, larger leaves, and stronger roots, which are very similar to the high-yield heterotic trait showed by the hybrid broccoli. In the present study, to uncover the roles of BolAHL16L in curd development of broccoli, different methods such as gene ectopic overexpression, gene targeted knock-out by CRISPR/Cas9, Chromatin Immunoprecipitation (ChIP), Yeast two hybrid and BiFC (Bimolecular Fluorescence Complementation) should be performed to identify the targeted genes regulated by BolAHL16L, and the key factors interacted with BolAHL16L. Following that the function of the genes and factors should be elucidated. The findings of the project should provide new insights into the function and molecular regulation mechanism of BolAHL16L in curd development, especially in the formation of high-yield trait of broccoli curd. Consequently, the investigations involved in the project are very important to deeply understand the nature basis of the formation and development of curd in broccoli.

花球是青花菜区别其它植物，由花序分生组织等构成的特化器官，也是其重要的产品器官。杂种优势利用是获得花球高产青花菜的最主要手段。但对青花菜花球发育，特别是花球高产性状形成机制的认识十分有限。项目前期获得一个与青花菜花球高产性状形成密切相关的转录因子BolAHL16L。研究表明BolAHL16L是AHL家族新成员，为首次报道。过表达BolAHL16L可使转基因拟南芥表现花枝及花蕾明显增多、叶片增大、根系更发达等类于高生物量杂种优势的性状表现。为此，项目拟综合运用基因过表达、CRISPR/Cas9基因敲除、染色体免疫共沉淀、酵母双杂交、双分子荧光互作等方法进一步对BolAHL16L调控的靶基因、与BolAHL16L发生互作的关键因子进行鉴定、克隆及功能解析，进而阐释BolAHL16L在青花菜花球发育中的功能及分子调控特征。研究对揭示青花菜花器官发育，特别是花球高产性状形成的分子基础具有重要意义。

在生殖发育过程中形成由花序分生组织等构成的花球，是青花菜区别于其他十字花科植物的最主要特征，花球也是青花菜最重要的产品器官，直接决定其经济价值。但对青花菜花球高产杂种优势性状形成的分子基础认识十分有限。项目前期通过基因表达谱分析、基因过表达功能分析初步证实AHL家族的一个新成员BolAHL16L，与青花菜花球高产杂种优势性状形成密切相关。在此基础上，本项目，综合运用基因过表达、CRISPR/Cas9 基因靶向敲除、酵母双杂交等研究方法对BolAHL16L在青花菜器官发育，特别是花球发育中的功能及调控机制进行了深入探究。结果表明，BolAHL16L正向调控青花菜器官发育，并主要通过对植株细胞数目的正向调控而在器官发育中发挥作用。进而对可能参与BolAHL16L调控通路的基因BolAp1A、BolCAL2S进行了克隆及功能探究，证实在拟南芥中过表达BolCAL2S可使转化株呈现出类于青花菜花球的花器官发育特征。进一步通过酵母双杂交文库筛选策略，筛选、鉴定并证实BolMYB21、BolPDI11、BolCOX11、BolPAP1、BolBIL4 五个蛋白与BolAHL16L存在强互作。此外，采用高通量转录组测序结合生物信息学分析，揭示了青花菜花球4个不同发育期的基因差异表达谱特征。获得了多个参与青花菜花球发育调控的新基因，并对其中的一个参与细胞数目调控的基因BolCNR6的表达特征及功能进行了深入探究，证实BolCNR6负调控青花菜器官发育，但其过表达可显著提高转化株对逆境的胁迫耐受。相关研究，对揭示AHL 类转录因子在青花菜器官发育，特别是花球发育中的功能及调控机制具有重要科学意义，同时对进一步揭示青花菜花球高产杂种优势性状形成的分子基础，开展花球高产青花菜分子育种具有重要价值。