玉米光周期敏感性主效QTL qDPS4候选基因ZmFKF1的克隆与功能研究

Photoperiod sensitivity is the main obstacle of the usage of tropical germplasms in temperate zone. So, identification, cloning and functional analysis of genes related to photoperiod sensitivity have significant value in theory and practice of agriculture. Based on the QTL analysis, the near-isogenic lines of QTL qDPS4 were constructed for the fine mapping, and then we obtained a candidate gene ZmFKF1 of a major QTL qDPS4 for photoperiod sensitivity. In order to understanding the function of ZmFKF1 in the photoperiod pathway，this study will mainly focus on three aspects: (1) cloning and analyze the sequences of CDS, promoter and genomic DNA of ZmFKF1，and analyze the expression pattern of ZmFKF1 under different photoperiodic induction in huangzao 4, CML288 and near-isogenic lines simultaneously; (2) construct two plasmids: one is knock-out of the ZmFKF1 by crispr-cas9 technology, the other is overexpress of the ZmFKF1, and validate the ZmFKF1 function by transgenic technology and phenotypic analysis of transgenic lines; (3) investigate the molecular workings of ZmFKF1 by some biochemical assays, including sub-cellular localization, pull-down, firefly luciferase complementation imaging assay. This study will prove new theories for comprehensive analytic molecular workings of photoperiod pathway in maize, and provide more information for abundance of maize germplasm and maize genetic improvement in China.

光周期敏感性是影响热带玉米种质利用的主要障碍，分离、克隆及对光周期敏感性相关基因的功能研究具有重要意义。本课题组在初步定位的基础上，构建了玉米第4染色体光周期敏感性主效QTL qDPS4的近等基因系，对其进行了精细定位，获得了候选基因ZmFKF1。本项目拟对该基因进行深入研究，主要包括：(1)克隆双亲及近等基因系ZmFKF1的CDS，启动子及基因组DNA序列，比较其序列差异，并分析该基因在不同光周期条件下的时空表达模式；(2)利用crispr-cas9系统构建ZmFKF1基因敲除载体，同时构建ZmFKF1过表达载体，通过遗传转化获得ZmFKF1基因敲除和过表达转基因株系，验证ZmFKF1功能；(3)利用亚细胞定位，pull-down，荧光素酶互补等技术探究ZmFKF1的分子作用机制。本研究将为全面解析玉米光周期调控开花的分子机制提供理论依据，对玉米遗传改良及拓宽我国玉米种质资源有重要意义。

光周期敏感性是影响热带玉米种质利用的主要障碍，分离、克隆及对光周期敏感性相关基因的功能研究具有重要意义。本研究在前期工作基础上，获得了玉米第4染色体光周期敏感性主效QTL qDPS4候选基因ZmFKF1，并对其进行了深入研究，确定了ZmFKF1的生物学功能，并初步说明了ZmFKF1的作用机制。主要研究结果包括：(1)确定了黄早4和CML288玉米中ZmFKF1的序列差异及时空表达差异，这些差异可能是造成黄早4和CML288光周期敏感性差异的原因；(2)通过分子生物学手段，获得了ZmFKF1基因编辑和过表达突变体。与野生型相较，ZmFKF1基因编辑植株表现出明显的晚花表型，且与玉米开花相关的关键基因ZmGI、conz1和ZmZCN8的表达量也明显下调，说明ZmFKF1位于这些基因的上游，正调控玉米开花过程。ZmFKF1过表达植株无明显表型；(3)亚细胞定位结果表明ZmFKF1、ZmCDF1和ZmGI定位在细胞核中。蛋白互作结果显示ZmCDF1与ZmGI互作。但是未能检测出ZmFKF1与ZmCDF1、ZmGI和ZmCO的互作。说明ZmFKF1发挥功能可能具有物种特异性。本研究结果为全面解析玉米光周期调控开花的分子机制提供了理论依据，对玉米遗传改良及拓宽我国玉米种质资源有重要意义。