ThABF转录因子在柽柳盐胁迫应答中的功能及调控机理研究

ABRE / ABF transcription factors are ABA responsive element binding proteins, involved in ABA signaling and stress responses in plants. In the previous study, we screened to obtain an ABF transcription factor，ThABF，an upstream regulatory protein of ThTIP associated with salt tolerance in Tamarix hispida. ThABF was induced by salt stress and improved salt tolerance of transgenic Arabidopsis thaliana, suggesting that it might be involved in the salt stress response of Tamarix hispida. On this basis, this project intends to study the function and regulatory mechanism of ThABF in response to salt stress in Tamarix hispida. The transgenic Tamarix plants that overexpression or inhibited expression of ThABF will be generated, and the changes of growth and physiological parameters in transgenic and non-transgenic Tamarix lines will be analyzed to determine the function in the salt stress response of Tamarix hispida. The cis-acting elements of ThABF promoter responding to salt stress will be identified. Based on the identified cis-elements, yeast one-hybrid is employed for investigation of the upstream regulators. ChIP-Seq and RNA-Seq will be combined to identify the target genes regulated by ThABF. Furthermore, the metabolic and physiological pathways these target genes involved will be analysed by integrating transcriptomics and metabonomics. The physiological mechanism of stress tolerance mediated by ThABF will be determined by analyzing the stress tolerance genes regulated by ThABF, and.physiological indexes will be monitored to further study physiological stress tolerance mediated by ThABF. These studies will be contributed in revealing the transcriptional regulatory networks of stress tolerance mediated by ThABF, to clarify the function and regulation mechanism of ThABF in response to salt stress of Tamarix hispida. The predict results will contribute to salt tolerance regulation mechanism of woody plants.

ABRE/ABF转录因子是ABA应答元件结合蛋白，参与植物的ABA信号转导和逆境胁迫应答。在前期的研究中，我们筛选到了柽柳耐盐基因ThTIP的一个上游调控蛋白（ThABF），其表达受盐胁迫诱导且提高转基因拟南芥耐盐性，推测其可能参与柽柳的盐胁迫应答。在此基础上，本项目拟对ThABF在柽柳盐胁迫应答中的功能及调控机理进行研究。将ThABF转入柽柳，分析过表达及抑制表达转基因株系耐盐能力，确定其耐盐功能。鉴定ThABF启动子中响应盐胁迫的顺式作用元件，通过酵母单杂交技术研究其上游的调控因子。利用ChIP-Seq技术、转录组学和代谢组学分析，并结合生理指标测定，鉴定盐胁迫下ThABF所调控的下游靶基因及靶基因参与的耐盐代谢途径和生理过程。通过以上研究解析ThABF参与的耐盐基因表达调控网络，最终阐明ThABF基因在柽柳盐胁迫应答中的功能及调控机理。预期研究结果为完善木本植物的耐盐机制具有重要意义

柽柳是一种具有很强抗旱、耐盐能力的盐生木本植物，是研究木本植物抗逆机理的理想材料。ABF转录因子是ABA应答元件结合蛋白，参与植物的ABA信号转导和逆境胁迫应答。在前期的研究中，我们在柽柳中筛选到了一个ABF转录因子（ThABF2），其基因表达受盐胁迫诱导且提高转基因拟南芥耐盐性，推测其可能参与柽柳的盐胁迫应答。本项目在ThABF2的上游调控因子、ThABF2调控的下游靶基因和识别的DNA序列以及ThABF2对柽柳的耐盐生理调节和代谢调控等方面系统研究ThABF2在柽柳盐胁迫应答中的功能及调控机理。. 利用启动子系列缺失的方法，鉴定了长度为242 bp（-216~-458 bp 区段）的启动子序列为ThABF2基因核心启动子区段。利用酵母单杂交技术获得了两个能够与ThABF2核心启动子区段结合的蛋白ThWRKY和ThMYC，这两个转录因子是ThABF2基因的上游调控因子。利用TF-Centered Y1H（以转录因子为中心的酵母单杂交）技术筛选ThABF2可能识别的DNA序列。结果显示，ThABF2转录因子可以特异性识别ACGTG(ABRE元件)和CGTAAT序列。通过农杆菌介导法将ThABF2基因转入柽柳，获得ThABF2过表达及RNAi沉默的柽柳转基因株系。盐胁迫下，ThABF2降低细胞ROS含量和细胞膜损伤程度，提高过表达转基因柽柳的耐盐能力，而RNAi植株耐盐性降低。转录组和代谢组联合分析的结果显示，ThABF2能调节柽柳体内腐胺的合成和积累，并调控腐胺代谢途径相关基因的表达。利用酵母单杂交、EMSA、ChIP-PCR和LUC实验进一步验证了ThABF2转录因子能够直接结合精氨酸脱羧酶基因ThADC启动子的ABRE元件并调控其表达。上述结果表明，ThABF2通过调控ThADC基因表达来参与腐胺的合成积累，提高抗氧化酶的活性，减少活性氧的积累，降低膜脂的过氧化损伤，从而提高柽柳对盐胁迫的耐受性。本项目的研究结果不仅丰富了木本植物抗逆机理的基础理论，同时也为林木抗逆分子设计育种提供了优秀的基因资源。