泛素连接酶CdF-box调控狗牙根抗寒性的分子机理

Bermudagrass (Cynodon dactylon) is a warm-season turfgrass and is adapted to cultivation in a wide range of climate condition. However,bermudagrass is relatively sensitive to cold stress which limited the extension in the Transition Zone. We previously characterized the response of bermudagrass to cold stress and used RNA seq technology to screen cold stress responsive genes. We found that 26 F-box genes were highly induced by the stress conditions. In this project, we will further explore the physiological and biochemical changes in bermudagrass after cold stress treatments and the detailed functions of F-box. We will clone F-box and DREB1 homolog genes from bermudagrass based on RNA seq data and/or RACE. The sequence differences among 3 bermudagrass cultivars and expression profiling of these genes under cold condition will be determined. For subcellular localization analysis, we will transform the according plasmids into tobacco leaf via transient transformation. Expression in plant tissues will be determined through GUS staining. Bermudagrass F-box and DREB1 genes will be transformed into model plant Arabidopsis and bermudagrass for functional analysis. Cold tolerance tests will be screened for transgenic Arabidopsis and bermudagrass plants. We will further screen interacting protein of F-box and DREB1 using bermudagrass cDNA library. We identified several proteins interacting with F-box which might play roles during DREB1 degradation through ubiquitination pathway. The detailed function of these interacting proteins will be further characterized. Finally, we will performed the experiment to determine how plant hormones improved cold tolerance of bermudagrass. This project will illuminate the detailed functions of F-box during the response of bermudagras to cold stress.

狗牙根是暖季型草坪草中坪用价值最高、应用最广的草种之一，缺点是其抗寒性差，限制了其应用。申请人前期对狗牙根低温转录组进行分析，挖掘了受低温影响的5个F-box基因。利用狗牙根低温cDNA文库筛选出了与CdF-box相互作用的蛋白。在此基础上，本项目将同源克隆抗寒性有明显差异的狗牙根品种中F-box和DREB1基因，分析其组织表达特异性和亚细胞定位；利用狗牙根和模式植物的转基因体系，解析狗牙根相关基因在低温应答中的功能；在酵母双杂筛选cDNA文库的基础上，利用Co-IP、BiFC及pull-down验证候选蛋白与F-box的相互作用关系；通过蛋白质体内和体外泛素化试验，阐述狗牙根F-box与候选蛋白的互作，从而介导相关蛋白如DREB1等的泛素化降解，调控狗牙根低温应答的机理；同时结合基因工程和外源物质处理的方法来提高狗牙根的抗寒性。

狗牙根(Cynodon dactylon)是暖季型草坪草“当家草种”，但是抗寒性差，在北方不能安全越冬，在华中和华东过渡带地区冬季休眠，一定程度上限制了其应用。本项目建立了狗牙根等草坪草抗寒评价的生理指标体系，阐述了蛋白质泛素化降解途径调控草坪草温度胁迫的机理，发现了ABA途径ABF转录因子调控DREB增强草坪草耐寒性的机制。项目结果表明，4℃低温和冻害处理严重抑制了狗牙根的生长，但是对冷季型高羊茅生长影响较小。狗牙根中脯氨酸和可溶性糖的积累显著低于高羊茅。转录组分析表明，低温处理后狗牙根中糖、纤维素、碳水化合物代谢途径、脱落酸和生长素等激素代谢途径富集。低温处理特异诱导了狗牙根DREB/CBF基因及上游调控因子的表达。启动子分析发现，FaDREB1A的启动子含有3个ABRE和2个LTR元件，而CdDREB1A的启动子有0个ABRE和1个LTR元件。在冷季型草中ABF与DREB1A启动子结合并调控后者的表达，增强了抗寒性。进一步转录组分析表明，大量参与蛋白质泛素化的F-box受到胁迫诱导。FaF-box和CdF-box转基因拟南芥植株具有显著增强的低温耐受性。分析发现狗牙根CdF-box启动子有4个ABRE响应元件。通过酵母双杂交筛选cDNA文库。获得了一批E3泛素连接酶相互作用的蛋白质，涉及温度胁迫及光合作用途径。通过多组学对狗牙根抗逆应答机理进行了探讨，根据相关系数和皮尔逊系数发现RNA-seq与i-TRAQ之间的相关性较低或呈中等相关。利用MapMAN软件分析了RNA-seq和i-TRAQ鉴定的差异基因和蛋白质中，糖异生/乙醛酸循环、光合作用、氨基酸代谢、氧化还原等途径显著富集。克隆了狗牙根CdABF2，亚细胞定位分析表明CdABF2主要在细胞核中表达。在拟南芥中异源表达CdABF2提高了萌发率和ABA敏感性。相关研究结果阐述了狗牙根应答低温胁迫的机理，为通过分子设计育种创制狗牙根新种质提供了理论基础和基因资源。