CBF介导干旱调控荔枝开花的作用机制研究

Litchi (Litchi chinensis) is economically important in southern China, where the accidental “warm weather” in winter always causes flowering obstacle due to inadequate chilling accumulation, leading to reduction of yield. The previous studies showed that drought stress prior to exposure to low temperature enhanced litchi flowering by reducing chilling requirement for litchi floral initiation, suggesting an important role of drought in litchi floral initiation. However, drought stress without followed low temperature treatment cannot promote litchi flowering. Plant flowering is regulated by multiple environmental cues, such as ambient temperature and soil water potential. Low temperature as a flowering promoter has been widely studied, whereas the effect of drought stress on plant flowering is still not fully understood, and thus it is necessary and important to clarify the effect and the molecular basis of drought on litchi initiation. In present project, potted ‘Feizixiao’ litchi plants will be treated with drought, low temperature and combination of drought and low temperature treatments to investigate the effect of drought stress on litchi flowering. The expression of CBF and well-known flowering-related genes in leaf tissues will be measured to identify the key genes in response to drought. The target genes of CBF will be identified by ChIP and the combination of CBF and the target genes will be confirmed by Yeast One-hybrid experiments. Taking all these together, this study potentially elucidates the involvemnt of CBF in promotion of litchi flowering by combination of drought and low temperature.

荔枝是华南地区极具经济价值的水果，亚热带地区出现的“暖冬”等异常天气，容易造成荔枝等果树成花需冷量不足，从而导致成花障碍。前期研究结果表明，低温诱导前给荔枝一定程度的干旱处理，可以降低荔枝成花对低温强度的要求，促进荔枝开花，但仅有干旱胁迫而无低温的外界条件，并不能促进荔枝开花，且干旱调控植物开花的分子机理仍然不清晰。因此，明确干旱对荔枝开花的调控机制及其分子机理极为重要。本项目以‘妃子笑’荔枝盆栽植株为材料，在开花诱导期进行干旱、低温，以及干旱结合低温处理，比较不同处理对荔枝开花时间的影响，通过Q-PCR等手段分析CBF及成花相关基因对不同处理的应答模式，筛选响应干旱的关键元件，并结合ChIP和酵母单杂等技术，鉴别与CBF转录因子互作的靶基因，以期阐明CBF转录因子介导的干旱调控荔枝开花的分子机理。

本研究对荔枝盆栽植株进行干旱、低温、以及干旱结合低温的处理，明确了干旱处理对荔枝开花的促进效应。对干旱处理的荔枝叶片进行转录组测序，结果显示LcCBF和LcWRKY等转录因子，以及开花相关基因LcMFT表达量被干旱上调，表明上述基因可能与荔枝成花相关。利用酵母单杂技术鉴定可知，LcCBF2、LcCBF5和LcWRKY均可以结合LcMFT基因的启动子。利用双荧光素酶系统检测可知，LcCBF2和LcCBF5 可正向调控LcMFT基因的转录水平，但LcWRKY对LcMFT基因的转录没有显著的调控效应。酵母双杂结果显示，LcCBF2、LcCBF5均可和LcWRKY发生蛋白互作，表明LcCBF2和LcCBF5可能通过与LcWRKY互作，共同调控下游LcMFT基因的转录。将LcCBF2、LcCBF5和LcMFT基因在拟南芥中过表达进行功能验证。结果显示，过表达LcCBF2和LcCBF5基因可显著提高拟南芥的抗旱能力，过表达LcMFT基因可显著提早拟南芥的开花时间。综上所述，干旱可上调LcCBF2/5和LcWRKY基因的表达，LcCBF2/5和LcWRKY通过蛋白互作，共同激活下游LcMFT基因的表达，从而促进荔枝开花。