DNA甲基化在牡丹内休眠解除中对生长发育及相关基因的调控

Anti-season forcing culture is one of the most important contents of tree peony （Paeonia suffruticosa Andr.）industry in China. The mechanism of endo-dormancy release in woody plants has been the hotspot in the recent years, which is also the theoretical basis for forcing culture in tree peony. The nature of dormancy release is to reinitiate growth from the status of growth cessation, so it has a great significance to understand the regulation mechanism of genes involved in growth & development. So far, extensive studies have been focused on the transcriptional level, but few information is available on epigenetic regulation. Based on our previous results,it indicated that DNA methylation invovled in the regulation of endo-dormancy release, and exogenous 5-aza C could promote dormancy release and acelerate sprouting in tree peony. Moreover, visible cell division and growth could also be observed in floral buds of tree peony during dormancy process. In this project, we aim to first study the regulation mechanism of DNA methylation on dormancy release. Dormant buds are treated with different chilling fulfilment and demethylating agent 5-aza C.The methylation levels and patterns, the dormancy status and growth & development characteristics at different treatments are investigated, and the temporal and spatial characters of DNA methylation in dormancy buds are examined by immunohistochemical study, to evaluate the co-relationship between DNA methylation, growth & development and dormancy release in tree peony. The growth & development candidate genes regulated by DNA methylation are screened by MSAP technology and subsequential bioinfaormatics analysis, and the methylation patterns are demonstrated by Southern blotting with restriction enzymes sensitive and insensitive to methylation. Then, they are identified according to their expression patterns during dormancy release induced by chilling. One or two growth & development related genes are then selected and cloned, and their functions are identified by over-expression in Arabidopsis. Subsequently, their expression characters under different treatments, are revealed by Northern blotting, Real-time PCR and RNA in situ hybridization. Moreover, the variation of methylation sites for the related genes during different treatments are evaluated by bisulfite methylation mapping. Taken together, all results aim to analyze the regulation of DNA methylation on the target growth & development processes and their related genes during dormancy release in tree peony, and they will help us to a closer understand the mechanism of dormancy release in tree peony, and provide guidances for artifically regulation florescence in tree peony forcing culture industry.

内休眠解除机理是牡丹主要产业- - 反季节催花的理论基础，其实质是从生长停滞状态重新启动生长的过程，因此生长发育相关基因是研究内休眠调控的关键候选之一。课题组前期研究发现：内休眠这一相对静止过程中，牡丹芽仍存在着细胞分裂与生长；外施5-aza C 可促进休眠解除，加快萌动生长，证明DNA甲基化参与了牡丹内休眠的解除。本项目拟研究低温和去甲基化试剂(5-azaC)处理条件下DNA甲基化水平和时空变化及其与生长发育状态的关系；并利用MSAP技术结合Southern杂交和基因表达模式筛选、鉴定甲基化调控的生长发育相关基因，选择克隆1-2个生长发育基因，并分析其功能；进一步分析生长发育相关基因在休眠解除中甲基化位点的变化及其时空表达模式。综上，解析DNA甲基化对牡丹休眠解除中生长发育及相关基因的调控机制，为实现人为调控花期提供理论基础。研究结果将成为内休眠机理研究的新增长点。

木本植物内休眠调控是落叶果树、花卉等植物反季节生产的理论基础，也是研究热点。最近的研究暗示着DNA甲基化参与了内休眠的调控，但一直缺乏可靠的证据和深入研究。本项目以此为切入点，分析了DNA甲基化对牡丹花芽内休眠及生长发育相关基因的调控。牡丹花芽休眠期间，仍存在组织学上的生长和发育，伴随着低温累积，花芽直径不断增加，表皮角质层和气孔数量增多，休眠的解除伴随着维管系统的建立。HPLC分析和免疫组化分析表明，低温累积降低了花芽中DNA甲基化水平，促进了花芽休眠解除，二者呈负相关性；5-azaC处理降低了牡丹花芽基因组DNA甲基化水平，提高了‘鲁荷红’萌动率和成花率，促进休眠解除；SAM处理提高了花芽内DNA甲基化水平，延长了‘鲁菏红’的休眠时间，证明了DNA低甲基化促进了牡丹花芽休眠解除。利用9个EcoRⅠ和8个HpaⅡ/MspⅠ引物组成共72对引物进行了MSAP分析，牡丹花芽甲基化程度高，以半甲基化状态为主，甲基化位点数占总位点的60%以上。‘鲁荷红’低温7 d、14 d、21 d和28 d去甲基化的位点数分别为211、280、353和513；过甲基化位点数分别为581、295、276和197，表明低温累积过程中去甲基化位点数逐渐增加，而过甲基化位点数逐渐下降，从而导致总甲基化位点数的下降。低温抑制了甲基转移酶基因PsMet、PsCMT，结构域重排甲基转移酶PsDRM基因的表达，诱导了去甲基化酶PsROS1基因的表达，从而调节了牡丹花芽中的甲基化水平。对MSAP获得的甲基化多态性片段进行了克隆，Q-PCR和亚硫酸盐测序验证，发现多个参与胁迫反应、代谢和生长发育相关基因受甲基化调控。5-azaC处理表现短期毒性效应和后期去甲基化作用，KEGG富集分析表明，其主要调控了DNA复制这一代谢通路。在此基础上，分析了PsMBD5、PsSPL、PSAP1等多个生长发育基因的表达调控和功能。以上研究结果已发表论文10篇，其中SCI论文3篇，培养研究生4名，获发明专利1项。