CYC2类同源基因Cyc2CL在菊花舌状花发育中的调控功能及作用机制

It has been revealed that the presence of the showy marginal florets in heterogamous capitula contributes to the pollination specialization, outcrossing rate and genetic diversity of the sunflower family, which is also the probable reason for the evolutionary success and rapid tribal radiation of the family. As the typical inflorescence structure of Chrysanthemum morifolium, capitulum is composed of two morphologically distinct florets; namely, ray florets and disc florets. Ray florets are ligulate and zygomorphic, with aborted stamens, and play an important role in attracting pollinators, which also has been shown to be associated with so many diverse flower types and colors in C. morifolium. The central disc florets with fertile pollens are radially symmetrical and hermaphroditic. However, the molecular mechanism regulating the development of zygomorphic ray florets remains unknown. . CYC2-like gene Cyc2CL was cloned from C. morifolium. It was found that Cyc2CL had two alternatively spliced transcripts: Cyc2CL-1 and Cyc2CL-2. There was no Cyc2CL-2 gene expression and minimal expression of Cyc2CL-1 in leaves and the apical buds during vegetative growth. Cyc2CL-2 expressed in the apical buds during flower differentiation. Both Cyc2CL-1 and Cyc2CL-2 had higher expression in ray florets than in disc florets. Cyc2CL-1 and Cyc2CL-2 were introduced into wild-type Arabidopsis, which showed that Cyc2CL-1 and Cyc2CL-2 can inhibit the development of stamens and petals in Arabidopsis. Cyc2CL-2 were introduced into wild-type chrysanthemums, which showed that Cyc2CL-2 can convert disc florets to ray florets in chrysanthemum. Therefore, it is indicated that Cyc2CL plays a key role during the process of stamen abortion in chrysanthemum ray florets.. Therefore, the project will aim to reveal the gene function and molecular mechanism of Cyc2CL during the developmental process of ray florets in chrysanthemum. We will use gene-editing technology and transgenic methods to obtain the overexpression and CRISPR/Cas9sgRNA mutant plants of Cyc2CL-1 and Cyc2CL-2, analyze the transcriptomic data of the overexpression and mutant plants combined with CHIP-seq technology to determine the downstream target genes of Cyc2CL-1 and Cyc2CL-2; verify the correctness of the downstream target genes by EMSA and yeast one hybrid assay, and explore if protein complexes were formed involving Cyc2CL-1 and Cyc2CL-2 and their co-regulators using pairwise protein–protein interaction assays.. This project will provide the first-hand knowledge for understanding the molecular mechanism regarding the formation of ray florets in chrysanthemum, and valuable genomic resources for candidate genes applicable for the breeding of novel varieties in chrysanthemum.

两侧对称雌性舌状花的出现，是菊科生殖生物学进化的重要结果，也是菊花形成丰富多彩的花型、花色的基础；然而，菊花舌状花形成的分子机制尚不清楚。申请者已经获得了菊花CYC2类基因Cyc2CL，发现花芽分化开始后Cyc2CL启动可变剪切，产生2个转录本（Cyc2CL-1和Cyc2CL-2）；将两个转录本在拟南芥中过表达，转基因株系花瓣缩短且雄蕊败育；将Cyc2CL-2导入菊花中过表达，转基因株系舌状花轮数增加、并出现了头状花序的管状花全部转变为舌状花的表型。本项目拟利用基因编辑和转基因技术，创造两个转录本的过表达和突变体株系，通过表型变异观察、组学和CHIP-seq分析，研究Cyc2CL-1和Cyc2CL-2的功能和下游靶基因，利用EMSA和酵母单杂验证其靶基因；利用酵母双杂解析其互作蛋白；最终探明Cyc2CL在菊花舌状花发育中的调控功能及作用机制，为菊花花型进化的分子机制研究提供重要理论基础。

本项目解析了菊花CYC2类基因Cyc2CL调控菊花舌状花形成的分子机制：Cyc2CL通过可变剪切机制转录两个mRNA：Cyc2CL-1和Cyc2CL-2；分别构建基因的过表达载体和CRISPR/Cas9sgRNA敲除表达载体转化菊花进行了基因功能研究，结果表明：Cyc2CL-1抑制菊花舌状花雄蕊的发育，Cyc2CL-2调控舌状花形成两侧对称性，Cyc2CL通过可变剪切机制调控了舌状花的两侧对称性和雄蕊败育性状的形成。亚细胞定位实验表明，Cyc2CL-1和Cyc2CL-2在细胞核内表达。酵母双杂交实验证明，Cyc2CL-1和Cyc2CL-2没有发生蛋白互作。以野生型拟南芥作为对照，分别对过表达拟南芥株系的花和叶片进行了转录组分析，探索了Cyc2CL-1和Cyc2CL-2的下游靶基因及其调控网络。Cyc2CL调控菊花舌状花形成的分子机制解析，为菊科头状花序的舌状花的进化形成提供了重要的理论依据。该项目建立了纳米磁珠介导花粉转基因技术，提高了转化效率，为菊花基因工程育种提供了有利工具。同时，该项目建立了利用CYC类基因创制雄蕊败育系的技术，为菊属及近缘种属雄性不育材料的创制提供了新思路和新方法，具有广阔的应用潜力。