梅花PmWRKYs转录因子参与冷胁迫应答的分子机理研究

Prunus mume is one of the traditional flowers in our country, which is restricted to open culture in North China by the dry-cold climate conditions in winter. In the previous study, we have identified three PmWRKY genes in Prunus mume, which were induced significantly in winter; in addition, the WRKY recognition site was found in the promoter regions of dehydrin gene PmLEAs, which are related to cold tolerance; so we speculate that transcription factor PmWRKYs may enhance the cold resistance of Prunus mume through up regulating the expressions of dehydrin gene PmLEAs. The variety ‘Sanlunyudie’ of Prunus mume will be used as experimental material in this study. Firstly, the expression patterns of PmWRKY genes will be analyzed under natural cooling process in winter and artificial low temperature treatment; secondly, transgene to model plants and genetic hybridization with the Arabidopsis mutant cbfs will be performed to verify the genes’ cold resistant functions and to construct their cold signal transduction pathway; lastly, the transcription factor activities of PmWRKYs and the binding activities to the promoters of dehydrin gene PmLEAs will be studied to verify the molecular mechanism that regulating dehydrin gene PmLEAs expressions by transcription factor PmWRKYs. Based on the above research, we expect to characterize the important cold resistant genes of Prunus mume, find new cold signal transduction pathway, and reveal the molecular mechanism of transcription factor PmWRKYs involved in cold stress response in Prunus mume.

梅花是我国的传统名花，在北方地区露地栽培受冬季干冷气候条件的限制。申请人在前期研究中发现3个PmWRKYs基因在梅花入冬后表达量显著升高，另外在梅花抗寒相关脱水素基因PmLEAs的启动子区域发现了WRKY识别位点，因此推测PmWRKYs转录因子可能通过激活脱水素基因PmLEAs的表达增强梅花的抗寒性。本研究以梅花‘三轮玉蝶’为实验材料，首先分析PmWRKYs基因在冬季自然降温过程和人工模拟低温条件处理下的表达模式；接着通过转基因模式植物，以及与拟南芥突变体cbfs的遗传杂交实验，验证基因抗寒功能，构建其低温信号转导通路；进一步分析PmWRKYs转录因子活性，以及与脱水素基因PmLEAs启动子的结合活性，验证转录因子PmWRKYs调控脱水素基因PmLEAs表达的分子机制。通过以上研究以期鉴定梅花重要的抗寒基因，发现新的低温信号转导通路，揭示梅花PmWRKYs转录因子参与冷胁迫应答的分子机理。

梅花原产我国，主要分布于长江流域、华南和西南等南方地区，在北方地区受冬季低温和干旱气候条件的限制，较难露地过冬。“南梅北移”，即让梅花的种植范围向北扩移，是梅花育种家坚持了几十年的育种目标。与传统杂交育种相比较，分子设计育种是以一种新兴的精准而高效的育种技术，但是需要夯实的理论研究和技术研发作支撑。本项目聚焦梅花低温应答转录因子PmWRKYs，对梅花基因组中的WRKY转录因子基因家族、PmWRKYs基因的低温诱导表达模式、转基因植株抗寒功能、PmWRKYs转录因子调控哪些靶基因表达等内容展开研究。我们的研究结果发现：梅花PmWRKY18、PmWRKY23和PmWRKY32基因受低温处理后表达水平明显升高，在冬季随气温先降低后升高的变化趋势，基因表达水平也呈先升高后降低的趋势；过量表达PmWRKY18的转基因烟草植株抗寒能力显著增强，且与对照比较根系更加发达；荧光定量实验表明PmWRKY18转基因烟草中NtCBFs的表达水平与对照比较显著升高，说明PmWRKY18可能是通过激活CBFs的表达来提高植株的抗寒能力；研究发现梅花抗寒功能基因PmLEA10的启动子序列中存在W-box元件，推测PmLEA10是PmWRKY18的靶基因，然而酵母单杂交实验表明PmWRKY18不能与PmLEA10的启动子结合，即PmLEA10不是转录因子PmWRKY18的直接靶基因。PmWRKY18作为梅花抗寒的正调控因子可以调节哪些下游基因的表达，其作用机制有待进一步研究。另外，酵母单杂交筛库发现梅花PmCBF3转录因子可以与PmLEA10的启动子结合调控基因表达，CBF转录因子在木本植物梅花中的作用机制与草本植物拟南芥是否存在差异，值得进一步研究。本研究鉴定了影响梅花抗寒的两个重要基因PmWRKY18和PmCBF3，为梅花抗寒机制研究奠定了理论基础，为梅花抗寒分子设计育种提供了优异基因储备。