LcNF-YB1调控中国石蒜成花的分子机制

Lycoris is known as red spider lily, which has many good ornamental traits, such as beautiful flower-like shape, rich flower colors and welcome bloom in summer when many flowers are dormant. It is an excellent summer bulbous flower with a high value for development and utilization. However, it has not been widely produced and used because of the long vegetative growth period of the bulbs (usually 3-5 years). Many studies have shown that the nuclear factor Y (NF-Y) gene families play an important regulatory role in flowering, but the molecular mechanism needs to be further clarified. In Lycoris chinensis, our previous studies found that the expression of LcNF-YB1 decreased with the growth of bulbs in vegetative growth, suggesting the negative regulation on transition from vegetative to reproductive growth. Therefore, in the present project, we intend to explore the gene function and regulatory mechanism of the LcNF-YB1 on flowering. The gene function will be verified through virus-induced gene silencing (VIGS) which will suppress the expression of LcNF-YB1 in Lycoris Chinese. The key genes in the downstream regulated by LcNF-YB1 will be analyzed by transcriptomic analysis. The yeast two-hybrid assay will be carried out to screen the interaction protein of LcNF-YB1, that will help to describe the interaction regulatory network of LcNF-YB1. Finally, the regulatory mechanism of LcNF-YB1 in flowering will be revealed at the level of transcription and translation. This project will provide genetic resources for the flowering regulation in Lycoris, and promote the innovation and utilization of germplasm, moreover, it will enrich the regulatory mechanism and theoretical basis of NF-Y gene families in plant flowering.

石蒜花形优美、花色丰富，且有花叶不相见的特性，是一类颇具开发潜力的夏季球根花卉。但石蒜开花需经历3-5年的营养生长期，成花转变耗时久，阻碍了其推广和应用。研究表明，核因子Y（NF-Y）对植物成花有重要的调控作用，但其分子机制有待进一步明确。申请人前期研究发现，中国石蒜LcNF-YB1基因表达量随种球年龄增加而降低，推测其可能负调控成花转变的进程。为研究其在成花调控中的功能及分子机制，本项目拟通过病毒介导的基因沉默（VIGS）抑制中国石蒜中LcNF-YB1表达，明确LcNF-YB1对成花的调控功能；利用转录组分析受LcNF-YB1调控的下游关键基因；通过酵母双杂交筛选LcNF-YB1的互作蛋白，解析其互作调控网络，最终揭示LcNF-YB1调控成花的分子机制。本项目研究不仅为石蒜成花调控提供基因资源，促进石蒜属植物的种质创新与利用，也为完善NF-Y调控植物成花的分子机理提供科学依据。

石蒜属（Lycoris）植物花型优美、花色丰富，花期7-9月，是一类颇具开发潜力的夏季球根花卉。但石蒜属植物开花需经历3-5年的营养生长期，成花转变耗时久，严重阻碍了其园林推广和应用。研究表明，NF-Y（NUCLEAR FACTOR Y）是植物发育和逆境响应的重要因子之一，对植物成花有重要的调控作用，但其分子机制有待进一步明确。该研究通过中国石蒜（L. chinensis）1-3年生营养生长的鳞茎、4年生营养生长和生殖生长的鳞茎分生组织转录组测序发现，部分NF-Y家族基因在不同样本中呈现差异表达，其中，LcNF-YB1随着种球年龄增加表达量降低，在生殖生长阶段表达量最低，推测其可能负调控成花转变的进程。进一步通过生物信息学分析、基因功能鉴定、下游调控网络解析、酵母双杂交等实验解析LcNF-YB1成花调控网络，得出以下结论：（1）LcNF-YB1全长1180 bp，编码194个氨基酸，具有保守的CBF（CCAAT box binding factor）结构域；（2）过表达LcNF-YB1的拟南芥株系开花推迟，通过VIGS沉默表达LcNF-YB1的中国石蒜株系花期提前，证明LcNF-YB1基因对开花的抑制作用；（3）LcNF-YB1亚细胞定位主要定位于细胞核中，但在细胞质内也有弱信号，推测存在核质穿梭的可能，转录活性实验表明LcNF-YB1不具有转录激活活性；（4）通过生物信息学筛选并验证了基因沉默中国石蒜植株中LcNF-YB1下游调控网络，包括：正调控基因可能为VIN3、NF-YA1/9、FLC基因等，负调控基因可能为FT、SPL14/15，以及开花启动因子等基因，明确了LcNF-YB1的转录调控机制；（5）对鳞茎分生组织不同发育阶段取样，提取RNA，构建中国石蒜鳞茎分生组织酵母文库，通过酵母双杂交技术从文库中筛选LcNF-YB1的互作蛋白，结果表明LcNF-YB1能够与开花调控路径中的关键因子LcCO5互作，明确了LcNF-YB1在翻译后和哪些蛋白互作共同调控中国石蒜的成花过程。从而阐明LcNF-YB1参与中国石蒜成花调控的分子机制，为石蒜属植物成花及花期调控的分子设计育种提供理论基础和优异基因储备。