miR399及其靶基因参与玉米开花期调控网络的机制研究

Maize (Z. mays ssp. mays) was domesticated from its wild ancestor, teosinte (Z. mays ssp. parviglumis), in the Balsas region of southwestern Mexico. Cultivated maize has been adapted to a wide geographical environment and is one of the most widely distributed crops in the world. As an adaptive trait, the maize flowering time can affect many agronomic traits directly or indirectly, such as yield and resistance. Therefore, it is particularly important to identify the genetic factors controlling the flowering time of maize and to dissect its molecular regulatory network. As a conservative group of microRNA in plants, miR399 was frequently involved in plant phosphorus stress response, but there are few reports on its involvement in plant flowering. In this study, by analyzing the spatial and temporal expression patterns of its family members, we identified a subset of miR399 members that might be involved in the flowering pathway of maize. The CRISPR/Cas9, STTM and overexpression technologies were used to obtain the corresponding mutants of miR399 members, STTM silencing and overexpression lines of miRNA family, respectively, to verify their effects on the flowering time in maize. At the same time, miR399 and its target genes were studied to construct the miR399-mediated regulation network in maize flowering pathway. Our study will provide a theoretical and practical basis for analyzing the domestication and adaptation process of maize and guiding the improvement of maize varieties.

玉米由分布于墨西哥南部巴尔萨斯流域的大刍草驯化而来。现代栽培玉米已经适应了极为广泛的地理环境，是世界上分布范围最广的作物之一。作为一个适应性性状，玉米开花期的早晚直接或间接影响着产量、抗性等众多农艺性状。因此，鉴定玉米开花期背后的遗传因子，解析其分子调控网络显得尤为重要。作为植物中相对保守的一类miRNA，miR399频繁参与了植物的磷胁迫反应，而关于其参与植物开花期的研究鲜有报道。本研究以玉米的miR399为研究对象，通过分析其家族成员的时空表达模式，筛选可能参与玉米开花途径的miR399成员；利用CRISPR/Cas9、STTM和过表达技术分别获得对应MIR399前体基因的突变体、miR399家族靶标沉默及过表达株系，验证其对玉米开花期的影响；同时结合生化、分子、遗传等实验手段构建miR399及其靶基因介导的玉米开花期调控网络，为解析玉米驯化适应过程、指导品种改良提供理论及实践基础。

营养素磷对植物生长发育至关重要。植物已经进化出多种策略来提高磷的吸收利用效率，以保护自己免受磷饥饿影响。然而，磷稳态与植物发育之间的串扰仍有待探索。本研究中，我们发现在玉米中过表达 microRNA399 会导致授粉后玉米早衰症状。敲除 miR399 靶基因 ZmPHO2 也会导致类似的早衰表型。有意思的是，我们玉米成花转变调节因子INDETERMINATE1 (ID1)可以通过直接结合 ZmMIR399 基因的启动子来抑制 ZmMIR399 基因的转录，减轻 miR399 对 ZmPHO2 的抑制，并最终有助于维持玉米体内的磷稳态。与由磷缺乏诱导表达的 ZmMIR399 基因不同，ID1 表达独立于外部磷水平干扰，表明 ID1 是磷稳态的自主调节因子。此外，我们发现 ZmPHO2 在玉米驯化和适应过程中受到选择，导致温带玉米比热带玉米对磷酸盐饥饿的反应更敏感。我们的研究揭示了 miR399-ZmPHO2 调节模块对磷胁迫的感知与 ID1 对植物发育的调节之间的直接功能联系。