菊花（Chrysanthemum morifolium）是世界四大切花之一，兼具较高的观赏价值和文化内涵。大多数菊花品种在秋季开花，在生产过程中需要经历高温炎热的夏季，并且时常伴随着干旱发生。干旱是影响植物最大的自然灾害之一，影响植物的生长发育以及繁殖。转录因子可以参与调控植物的生理反应、生长发育和非生物胁迫诱导的信号传导。本研究在前期发现菊花CmBBX24.1参与干旱胁迫调控的基础上，旨在深入探究其干旱胁迫调控的机理。研究结果如下：

1. 构建CmBBX24.1的过表达载体pORE-R4-CmBBX24.1，通过农杆菌侵染菊花叶盘转化获得CmBBX24.1的过表达转基因株系，通过气孔导度测定发现其具有更小的宽长比。在自然条件下CmBBX24.1转基因株系不具有开花差异，经过干旱处理后，融合抑制子基因沉默株系pSRDX-CmBBX24.1与野生型相比，表现出提前开花，而超表达没有显著差异。对CmBBX24.1融合抑制子基因沉默株系进行RNA-Seq分析，发现一个钙调蛋白样蛋白基因CmCML20以及通过调节气孔进而负调控耐旱性的AGL16同源基因上调表达，CmBBX24.1可能通过ROS途径以及Ca2+信号转导调控菊花的干旱耐受性；在成花转变期CmBBX24.1可能通过调控自主途径相关基因和MADS家族开花相关基因的表达，以及代谢途径基因响应和其他胁迫相关调控因子来调控开花。

2. 为了进一步明确CmBBX24.1翻译水平的调控机制，利用CmBBX24.1为诱饵进行酵母双杂交筛库，筛选到NF-YC亚家族基因NF-YC9，其ORF全长768 bp，编码255个氨基酸残基。转录激活和亚细胞定位实验证明了CmNF-YC9具有转录激活活性，且在细胞的核、膜中均有定位。通过酵母双杂交、烟草荧光素酶（LUC）、洋葱双分子荧光互补（BiFC）和体外pull-down等实验证明CmBBX24.1与CmNF-YC9互作。系统发育树分析显示CmNF-YC9与CsNF-YC亚基存在多条相似的蛋白序列，且均受到干旱诱导表达。组织表达模式分析表明，CmNF-YC9与同源CsNF-YCs在管状花中的表达量较高。通过农杆菌介导的叶盘侵染法获得CmNF-YC9稳定转化的转基因株系，结果表明过表达pORE-R4-CmNF-YC9转基因植株表现出更强的耐旱能力，存活率显著高于野生型，而融合抑制子基因沉默株系pSRDX-CmNF-YC9则相反。这说明CmBBX24.1与CmNF-YC9协同调控菊花的耐旱性。

Chrysanthemum morifolium is one of the four major cut flower in the world, with both high ornamental value and cultural connotation. Since most chrysanthemum varieties bloom in autumn and suffer form high-temperature summer and often occur with drought during the production process. Drought is one of the most impactful natural disasters, often affecting plant growth and development, as well as fertility and reproduction. Transcription factors can participate in the regulation of physiological responses, growth and development, and signaling pathways induced by abiotic stress in plants. In our previous study, we found CmBBX24.1 involved in drought stress. This study aimed to further explore the molecular regulation mechanism of CmBBX24.1 in response to drought and drought-mediated flowering function. The results are as follows:

1. An overexpression vector of CmBBX24.1, pORE-R4-CmBBX24.1, was constructed to obtainoverexpression transgenic lines by agrobacterium-mediated transformation with chrysanthemum leaf discs, and it was found that it had a smaller stomatal width to length ratio. At the same time, it was found there was no flowering difference between CmBBX24.1 transgenic lines under natural condition. After drought treatment, the chimeric repressor gene-silencing lines pSRDX-CmBBX24.1 showed early flowering compared with wild type, while there was no significant difference between overexpression lines and wild type plants. RNA-Seq analysis of the pSRDX-CmBBX24.1 lines revealed that calmodulin-like protein gene CmCML20 and AGL16 homolog gene, which negatively regulated drought tolerance by regulating stomatal movement, were up-regulated, and that CmBBX24.1 regulated drought tolerance in chrysanthemum under drought stress through the ROS pathway and Ca2+ signaling. CmBBX24.1 participated in regulating flowering by regulating multiple genes expression, including autonomous pathway genes, multiple MADS family flowering-related genes, metabolic pathway genes and other stress-related regulators.

2. In order to further clarify the molecular mechanism at the translation level, the NF-YC subfamily gene NF-YC9 was screened with yeast two hybrid using CmBBX24.1 as bait. CmNF-YC9 was cloned from Chrysanthemum 'Jinba', with an ORF length of 768 bp and encoding 255 amino acid residues. Transcriptional activation and subcellular localization experiments proved that CmNF-YC9 had transcriptional activation activity and existed in the nucleus and membrane of cells. Experiments such as yeast two hybrid, luciferase assay (LUC) in tobacco, bimolecular fluorescence complementation (BiFC) in onion assay and pull-down in vitro proved that CmBBX24.1 interacted with CmNF-YC9. Phylogenetic tree analysis showed that CmNF-YC9 and CsNF-YC subunits shared multiple similar protein sequences and all of them were regulated by drought. Tissue expression pattern analysis showed that CmNF-YC9 and homologous CsNF-YCs had higher expression in disc flowers. CmNF-YC9 transgenic lines were obtained by agrobacterium-mediated transformation with leaf discs. Overexpression CmNF-YC9 transgenic plants showed greater drought tolerance with significantly higher survival rates than wild type, while the gene silencing lines pSRDX-CmNF-YC9 were sensitive to drought stress than wild type. These results indicated CmBBX24.1 interacted with CmNF-YC9 to regulate drought response in chrysanthemum.

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