miR159及其靶基因GAMYB调控春兰开花的作用机制研究

Cymbidium goeringii is one of the most widely distributed, abundant and commonly cultivated orchids in China, with extremely high ornamental and economic value. However, the low differentiation rate of flower bud of Cymbidium goeringii severely limits its commodity value and industrial scale. miR159 (microRNA159) is specifically induced in the transformation from vegetative growth to reproductive growth of Cymbidium goeringii. Further degradation and sequencing revealed that the target gene GAMYB of miR159 is the key factor of the gibberellin signaling pathway. This study first analyzed the splice site of miR159 in GAMYB of Cymbidium goeringii, and the relationship between the related physiological indexes and the expression of miR159-GAMYB during its flowering. Then, the regulation of miR159 in the related physiological indexes and key genes in the flowering induction of Cymbidium goeringii was investigated by the transient expression of virus-mediated miR159 in the flower bud of Cymbidium goeringii. Finally, the downstream genes of GAMYB were screened and their regulatory relationships were verified. With miR159 as the breakthrough point, this study reveals the regulating mechanism of miR159-GAMYB-mediated gibberellin signaling pathway in the flowering of Cymbidium goeringii by physiology, genetics and molecular biology, providing a theoretical basis for the regulation of the flowering of Cymbidium goeringii.

春兰是我国分布最广、资源最丰富、栽培品种最多的兰花之一，具有极高的观赏和经济价值。但是，春兰花芽的低分化率严重限制了春兰的商品价值与产业规模。miR159（microRNA159）在春兰营养生长向生殖生长转化的过程中，受到特异诱导。进一步降解组测序得到了miR159的靶基因是赤霉素信号途径的关键因子GAMYB。本项目首先分析春兰miR159在GAMYB的剪切位点，解析春兰成花过程中相关生理指标与miR159-GAMYB表达水平的关系。其次，通过病毒介导的miR159在春兰花芽中的瞬时表达，研究miR159对春兰开花诱导相关生理指标和关键基因的调控。最后，筛选GAMYB的下游基因，并验证它们的调控关系。本研究以miR159为切入点，利用生理、遗传和分子生物学等研究手段，揭示miR159-GAMYB介导的赤霉素信号途径参与春兰开花的调控机制，为春兰花期调控提供理论依据。

春兰（Cymbidium goeringii）是一类地生兰，植株纤柔，花色清雅，多具幽香，品种繁多，极富观赏及经济价值。春兰苗期较长，花期短，花芽分化率低，开花时间与市场需求高峰不一致，极大制约了兰花的商品价值和产业发展。因此，研究春兰生长发育机制具有重要意义。miR159是植物microRNA中较为保守的古老家族，可参与多种生理过程。本研究克隆并构建了春兰miR159的过表达载体并异源转化至拟南芥，通过遗传学、形态学、细胞学、分子生物学及生物信息学等研究手段，探究了春兰生长发育相关miR159的功能。研究发现春兰miR159结构保守，功能强大：生物信息学分析表明，cgo-MIR159的结构具有一定的保守性，进化性较高，其中包含STRE、TATA-box、TATC-box、CAAT-box、CCTC等元件；cgo-miR159成熟体的结构高度保守，但遗传距离与多数植物的miR159相距较远；cgo-miR159靶基因多为MYB类基因，MYB靶标的功能可富集于多种生物学过程。荧光定量实验结果表明，cgo-miR159在春兰营养生长向生殖生长转变、花芽和叶片的发育、ABA胁迫应答等过程中，表达水平均有较大变化。同时，春兰miR159在模式植物的生长发育过程中发挥重要作用：遗传学、形态学与细胞学实验表明，cgo-miR159的异源过表达可缩短拟南芥的寿命，提前花果期；能够影响拟南芥幼苗和营养器官的发育、雄蕊、花粉、果实和种子的生长发育。