油菜素甾醇调控梨花芽休眠解除过程的机理研究

The process of endodormancy release of fruit trees is regulated by the dynamic balance of multiple phythormones. To date, much effort has been made to unveil the functions of ABA and GA on the endormancy release process while less attentions have been paid on other phytohormones. Brassinosteroid (BR) is a kind of phytohormone that promotes plant growth. In model plant Arabidopsis, it is clear that BR is involved in the seed dormancy breaking and germination through the analogous effect with GA and the antagonistic effects of ABA. However, the function of BR in endodormancy release of tree buds has not been well studied. Our preliminary results showed that the expression of a key enzyme in BR biosynthesis, CYP85A2, was up-regulated during the chilling accumulation in the pear flower bud, suggesting the involvement of BR in the endormancy release. In this project, typical pear cultivars with different dormant characteristics will be used to study the function of BR on endodormancy release process. The changes of endodormancy release process and the endogenous BR levels will be recorded after exogenous BR or BR inhibitor brassinazole treatment, during which the gene expression of key candidate genes participating in BR biosynthesis and signal transduction pathway will be determined. In addition, the crosstalk between BR pathway and ABA,GA pathways will also be analyzed. Finally, the function of BR on endodormancy release will be confirmed using the transgenic European pears harboring the overexpression or gene silencing cassette for the key BR biosynthesis enzyme CYP85A2. This project will enrich the theory of molecular mechanism of pear endodormancy release, provide the theoretical basis for breeding new pear cultivars with low chilling requirement and develop novel technology for artificially breaking endodormancy.

果树休眠的解除过程受到多种內源激素的动态变化及平衡调控。迄今为止的研究主要集中在ABA与GA上，而对其他种类激素研究较少。油菜素甾醇（BR）是一类促进生长的激素，也能够促进种子休眠的打破并促进萌发，其作用与GA同向而与ABA相反。本人前期工作发现，BR生物合成过程的关键酶CYP85A2在砂梨花芽低温积累过程中上调表达，暗示其可能参与了梨的休眠解除过程。本研究拟以具有不同需冷量的梨品种为试材，通过观察外施BR或BR合成抑制剂对休眠解除的影响来分析BR在此过程中发挥的作用。进而通过生理指标的测定，相关基因的克隆及表达分析，BR与ABA,GA代谢与信号转导过程的互作分析以及转基因验证等多个角度探索BR参与调控休眠解除过程的生理与分子生物学机制。预期研究成果将有助于阐明梨花芽休眠解除过程的分子调控网络，为利用分子技术手段培育低需冷量梨新品种和开发新型破眠剂提供理论支持。

梨是我国重要的落叶果树，芽内休眠能否顺利解除直接影响果实的产量和品质。一般认为，GA与ABA是调控休眠过程的重要激素，但是其作用机制以及所受到的调控模式尚未得到阐释。蔷薇科果树中的Dormancy Associated MADS-box (DAM)基因是调控休眠过程的关键基因，抑制芽的萌发过程，但其所受到的调控尚未得到详细研究。本项目研究了油菜素内酯（BR）对梨花芽休眠解除的效果并分析了其作用机理，所得结果如下：.1.BR处理结果显示，BR处理能够替代部分低温积累，促进花芽解除休眠。在此过程中，BR处理显著抑制ABA的合并促进了ABA的降解，同时通过与DELLA蛋白互作参与GA信号转导过程。.2.ABA是诱导梨进入休眠过程的关键激素，ABA信号合成与转导相关基因在此过程中发挥了重要作用，然而梨ABI5无法直接激活DAM基因的表达，但梨中的CBF基因的表达均受到ABA的调控，而CBF能够转录激活DAM基因。.3.基因家族分析结果显示，梨中存在4个CBF基因，这些基因在休眠过程中表达模式存在较大差异。4个CBF基因选择性激活梨基因组中的3个DAM基因的表达，从而调控低温诱导的休眠过程。.4.GA处理能够部分代替低温积累量，达到提前打破休眠的效果。在此过程中，我们筛选获得了GAST1基因，该基因在休眠解除时大量上调，且与花芽内的GA含量变化一致。转基因试验结果显示，GAST1基因能够减弱拟南芥种子的休眠，促进萌发。对梨GAST基因家族进行分析结果显示，其他多个GAST基因也在休眠过程中差异表达，可能参与了梨芽休眠过程的调控。.综上所述，本研究发现BR有助于促进休眠的解除过程，为我们开发新型破眠剂提供了思路。此外，本项目还初步研究了ABA与GA在休眠建立与解除过程中的作用机制，为进一步完善梨休眠的调控网络打下了坚实的基础。