ABA调控冬小麦糖代谢应答低温胁迫的生理分子机制研究

Dongnongdongmai 1(dn 1) was the first freezing-tolerant(resistant to low temperature of -30℃)winter wheat that can live through the winter in extreme frigid region in Heilongjiang Province.The reviving rate was greater than 85% which was considered to be a precious material for cold research. Sugar metabolism in plant took part in the response to cold stress. Our previous study indicated that low temperature could increase the sugar content and the activity of related metabolism enzymes in winter wheat. In addition, all the changes were affected by exogenous ABA, but the mechanism was not cleared.dn1 was used as materials, after dealt with ABA, the tillering nodes were sampled under natural cooling condition in fields. Sugar content and the expression pattern of enzyme genes will be tested using the physiological, biochemical and molecular levels methods., and then the physiological mechanisms of ABA regulating sugar metabolism in winter wheat were cleared. Based on these, the key enzyme genes will be cloned, and their functions be verified. Two-dimensional electrophoresis will be used to acquire differential proteins which are related to ABA regulation. After bioinformatic analysis, the functions, metabolic pathways, signaling pathways and protein - protein interaction networks involved will be determined. The physiological and molecular mechanisms of ABA regulation of sugar metabolism in winter wheat in the response of low temperature stress are revealed by integrating the physiological mechanisms, gene analysis and proteomics forecast. Our project is featured by researching on winter wheat in extreme frigid region. The results could supply new ideas for cold-resistance molecular breeding and cultivation, and rich cold-resistance genetic resources.

东农冬麦1号(dn1) 是我国首例能在黑龙江省高寒地区越冬的强抗寒(可耐-30℃低温)冬小麦 (返青率＞85%),是耐寒研究珍贵材料。植物的糖代谢参与低温胁迫应答。我们前期研究发现,低温可促进冬小麦糖含量及代谢相关酶活性增加,且这些变化受外源ABA影响可增强抗寒性,但作用机制尚不清楚。本项目以dn1为材料,ABA处理后,大田自然降温条件下取样分蘖节,采用生理生化及分子生物学手段,检测糖含量及代谢关键酶活性与酶基因表达情况,明确ABA调控冬小麦糖代谢的生理机制；在此基础上,克隆关键酶基因,验证功能；同时通过双向电泳和质谱技术获得与ABA调控相关的差异蛋白,生物信息学分析,确定其功能和参与的代谢途径、信号途径及蛋白互作网络。综合糖代谢生理机制、基因分析和蛋白组学预测,揭示ABA调控冬小麦糖代谢应答低温胁迫的生理分子机制。项目有高寒地区冬麦研究特色,结果可为抗寒分子育种及栽培调控提供新思路及基因

项目以强抗寒冬小麦品种Dn1为材料，适宜浓度ABA处理后,大田自然降温(5℃、-10℃和-25℃)条件下取样分蘖节，运用生理生化、分子生物学和生物信息学等手段，探讨ABA调控冬小麦糖代谢应答低温胁迫的生理分子机制。（1）分别建立了三个温度下的蛋白质组学、 代谢组学、miRNA和lncRNA测序数据库。（2）蛋白组学分析筛选到糖代谢相关蛋白10种，主要为参与蔗糖代谢、EMP、TCA、PPP等途径的关键酶。（3）代谢组学分析筛选到糖代谢相关产物20种，主要参与蔗糖代谢、EMP、TCA、PPP和糖异生途径。（4）miRNA数据分析筛选到参与果聚糖和淀粉代谢通路的miRNA10个、靶基因11个；外源ABA可通过调节miR5049-3p和mi1120a影响其靶基因6PGL和FBA的表达量，进而介导糖代谢响应低温胁迫。（5）构建了冬小麦抗寒相关lncRNA-miRNAs-靶mRNAs互作关系图，筛选并验证了三种lncRNA可作为ceRNA调控Tae-miR398与其靶基因应答低温胁迫。（6）低温胁迫下6个转录因子参与低温应答，外源ABA正调控TabZIP1表达，利于提高小麦抗寒性。（7）ABA可通过正调控TaCBP3、TaCBP5、TaNPQ4表达量变化、相关光合参数、抗氧化酶活性等变化，以及正调控PPT途径和蔗糖代谢的关键调节酶活性及其基因表达参与冬小麦抗寒应答。-10℃是冬小麦启动抗寒机制的关键温度点。综上糖代谢生理机制、基因分析、蛋白组学和代谢组学预测及相关检测验证,初步揭示了ABA调控冬小麦糖代谢应答低温胁迫的生理分子机制，研究结果对于深入解析作物抗寒机制及抗寒分子育种与栽培调控管理提供了科学依据，具有重要的理论和实践意义。项目资助发表论文共14篇，其中SCI收录3篇，中文核心期刊11篇，待发表SCI收录文章3篇。培养研究生18人，其中已毕业13人（含博士1人），在研5人（含博士2人）。