枳β-淀粉酶基因PtrBAM1低温应答分子机制及调控网络解析

PtrBAM1 of Poncirus trifoliata plays a role in starch degradation, and accumulation of soluble sugars and functions in cold tolerance. However, the molecular mechanisms underlying induction of PtrBAM1 by cold and the regulatory network remain unclear at this point. In the current project, critical cis-acting elements required for cold response of PtrBAM1 will be identified in the promoter of PtrBAM1. Meanwhile, methylation status and patterns of the coding region and promoter of PtrBAM1 will be analyzed. These work will help to elucidate the molecular mechanisms underlying cold response of PtrBAM1. Relevant transcriptional factors (TFs) that bind with the identified cis-acting elements will be isolated. Specific binding of the TFs to PtrBAM1 promoter will be verified. Meanwhile, regulation of PtrBAM1 expression and accumulation of soluble sugars by the TFs will be clarified, while the roles of TFs in cold tolerance will be dissected. In addition, proteins, particularly protein kinases, interacting with the TFs will be explored. Interaction between the interacting proteins (IPs) and the TFs will be verified. Furthermore, implication of the IPs in the TFs-mediated regulation of PtrBAM1 expression and accumulation of soluble sugars under cold stress will be assessed. The current project will provide clues for illustrating the molecular mechanisms and regulatory network underlying the cold response of PtrBAM1, and understanding the theoretical basis on cold-induced soluble sugar accumulation; all these will further our knowledge on the cold response of plants. On the other hand, potential elite genes that can be used for stress tolerance-oriented genetic engineering of citrus will be exploited, manifesting the scientific significance and potential application of this project.

枳（Poncirus trifoliata）β-淀粉酶基因PtrBAM1在淀粉分解、可溶性糖积累中起作用并具有抗寒功能，但它响应低温的分子机制及调控网络不明晰。本项目将鉴定PtrBAM1启动子低温应答顺式作用元件，分析PtrBAM1编码区和启动子甲基化模式，以揭示PtrBAM1响应低温的分子机制。在此基础上，分离与低温应答元件结合的转录因子，验证转录因子与PtrBAM1启动子特异性结合，阐明转录因子在调控PtrBAM1表达及可溶性糖积累中的作用，解析其抗寒功能。筛选转录因子的互作蛋白（蛋白激酶），验证互作蛋白与转录因子的互作，并鉴定互作蛋白在转录因子调控PtrBAM1表达和可溶性糖积累中的功能。本项目一方面将揭示PtrBAM1响应低温的分子机制和调控网络，阐明低温下可溶性糖积累的理论基础，丰富低温应答的理论认识；另一方面，为柑橘抗逆分子改良提供基因资源，因而具有重要科学意义和潜在应用价值。

本项目揭示了PtrBAM1亚细胞定位于叶绿体，在烟草中超表达PtrBAM1后显著提高了烟草的抗寒性。其启动子包含多种转录因子的结合元件；通过逆境、ABA及ABA与抵制剂处理分析表明胁迫下可溶性糖的积累依赖于ABA。进一步研究了能调控PtrBAM1表达的转录因子，酵母单杂交实验证明三个CBF基因（CBF2、CBF3、CBF 4）以及一个ABF基因（ABF4）均可与PtrBAM1启动子互作，双荧光素酶实验发现CBF3激活PtrBAM1启动子最显著；分离了一个可能负调控PtrBAM1的MYB基因（MYB3）；研究表明，转录因子PtrERF109和PtrERF10定位于细胞核且具有转录激活活性，它们均正调控抗寒性。转录组分析表明，PtrERF109和PtrERF10调控了大量糖代谢途径的基因，可能是低温下糖积累的重要调控因子。利用酵母双杂交和Pull-down表明蛋白激酶SnRK2.4和SnRK2.4能与ABF2在体内和体外互作。此外，发掘了柑橘甲基化相关基因，并分析了它们在低温下的表达模式，发现MET1受诱导最为显著，表明甲基化可能参与低温应答。项目的开展为揭示PtrBAM1低温应答的转录调控网络奠定了基础，鉴定的抗寒基因为今后开展基因工程提供了重要的基因资源。共发表SCI论文6篇，申请国家发明专利2项，获得个人奖项和荣誉称号多项，培养研究生8人，其中博士生6人，硕士生2人。