DNA甲基化在ABA调控草莓休眠诱导中的作用机制研究

Strawberry is a perennial herb, through a dormant state responses to cold in winter. Strawberry fruit use being uneasy of storing up, that should be regulated dormant and growth cycle by facility cultivation to prolong the fresh supply. Many factors are involved indormancy regulation, which include environmental factors, such as photoperiod and temperature Until now, the research actuality about strawberry dormant rests on environmental aspect and physiological metabolism, but lack of deep research on molecular mechanism, which constrains the development of Chinese strawberry industry. Through the preliminary experiments, we get to the conclusion that dormancy is a gradual process induced by the interactions of environmental factors which triggers the biochemical changes such as the change of expression of ABA. DNA methylation was shown a dynamic change during induction of dormancy, but the molecular mechanisms of DNA methylation control dormancy is unclear. further research is required to investigate the methylation of specific genes involved in dormancy regulation combined with the study of responses to exogenous phytohormones in different cultivars showing different dormancy induction characteristics. This project research analyzed the strawberry growth and ABA（Abscisic acid）content variation effects of DNA methylation changes, studied the DNA methylation changes and methyltransferase FaDRM1、FaROS1 gene expression level effects of ABA changes in the process of induced dormancy, expounded the role of DNA methylation in the dormant regulation. ABA synthesized key enzyme NCED1 and dehydrin protein gene were coloned, and detected by real-time quantitative PCR, methylation status of promoter by methylation specific locus MSP, proclaimed the mechanism of DNA methylation in ABA regulation dormancy. This project has the positive significance to dormant theory and application in production of fruits and vegetables.

草莓是多年生草本植物，通过休眠应对低温环境。设施栽培调控草莓的休眠与生长期，可延长鲜果供应时间，但尚未实现鲜果周年供应。目前国内外对草莓休眠的研究多集中在环境因素和生理代谢水平，对其分子机制缺乏深入研究，制约了设施栽培发展。前期研究发现，DNA甲基化在草莓休眠中呈现动态变化，但其在调控休眠中的作用机制尚不明确。本项目拟分析草莓休眠诱导过程中DNA甲基化水平改变对草莓休眠和ABA(Abscisic acid)含量变化的影响；研究诱导休眠过程中ABA水平改变对DNA甲基化和甲基转移酶FaDRM1、FaROS1等基因表达变化的影响，探讨DNA甲基化和ABA在调控休眠中的作用关系；克隆ABA合成关键酶NCED1和脱水素蛋白基因及其启动子，分析基因表达水平和启动子甲基化状态，揭示DNA甲基化在ABA调控草莓休眠中的分子机制。本研究为草莓休眠机制的深入阐述与完善提供理论依据。

莓植株进入休眠后，植物体的外表停止生长，但内部生理代谢活动并未停止，体内的营养物质、酶的活性以及内源激素都在发生一系列的积极的变化。本课题旨在分析草莓休眠过程中DNA甲基化和ABA(Abscisic acid) 水平改变对草莓休眠的影响，以及DNA甲基化和ABA在调控休眠中的相互作用关系等。研究结果表明，草莓在受到休眠诱导后，新叶叶片中ABA和DNA甲基化水平都增加，且ABA变化和总DNA甲基化水平变化同步。草莓解除休眠后甲基化水平显著下降，从这种甲基化水平的下降，有利于参与植株代谢过程的一些基因表达，促使植株休眠的解除。用浓度为25mg/L ABA对草莓叶片进行喷施处理，草莓叶片在ABA处理之后，基因组总DNA甲基化水平上升，ABA的水平影响基因组DNA总甲基化水平。克隆了植物休眠相关基因，并研究这些基因在草莓休眠不同时期的表达情况，研究不同休眠时期FaDRM1、FaROS1、FaNCED1、FaPIN等基因表达变化，发现在不同休眠时期一些基因表达会显著上调或者下调。结果表明，环境变化首先引起ABA作出反应，ABA变化进一步调控DNA甲基化、染色质重组等表观遗传变化，表观遗传与ABA参与的其它信号通路共同调控休眠。除了这条通路中各因素影响休眠之外，其它植物激素、糖类、氧化还原酶等各种酶类和其它一些信号通路也参与调控休眠，各个因素之间的协同作用共同调控休眠。同时利用草莓休眠的生理特点，研究通过成品苗冷藏或假植达到自由控制成熟期，实现草莓鲜果周年供应。结果显示，草莓苗适宜的冷藏温度为0℃左右，冷藏期间，温度应严格控制并稳定在0℃左右。提供出冷藏草莓种苗出库后的定植方法，探索了延迟栽培冷藏种苗出库后的定植和田间管理方法，提高了种苗成活率和果实产量。项目研究为植物休眠机制的深入阐述与完善，设施栽培发展提供科学依据。