镉胁迫下巨大芽孢杆菌NCT-2诱导龙葵水杨酸合成的调控机制

The strengthen measure of microorganisms on phytoextraction is effective for remediation of heavy metal contaminated soils. Beneficial microbes can alleviate the toxicity of heavy metals by inducing synthesis of salicylic acid (SA) in plant, however, the inducing factors and regulatory mechanisms are not clear yet. It was shown that under cadmium stress, the secretory protein of Bacillus megaterium NCT-2 was essential for inducing SA synthesis in S. nigrum L., but the types of protein and its regulatory mechanism need to be further studied. For B. megaterium NCT-2, the different proteins will be systematically investigated by secretome under cadmium stress. Then the protein types will be analyzed by bioinformatics. The shuffling genes deletion mutants will be constructed to identify the types of proteins which could induce the synthesis of SA . After that, the regulatory effects of secretory proteins on SA synthase and its key transcription factors will be investigated through GST pull-down and EMSA. Finally, the effective secretory proteins will be overexpressioned in B. megaterium NCT-2 and S. nigrum L. to promote the synthesis of SA. The general applicability of strengthen measure of microbial secretory proteins on cadmium phytoextraction will be understand, as well as the induction mechanism of SA synthesis. The study will provide theoretical basis for the strengthen measure of beneficial microbes, such as Bacillus, which are effective on remediation of heavy metal contaminated soils，and provide technical support for for improving cadmium phytoextraction efficiency.

利用微生物强化植物萃取是修复土壤重金属污染的有效途径。有益微生物可通过诱导植物合成水杨酸(SA)来缓解重金属毒害，然而诱导因子及其调控机制尚不明确。申请者在前期研究中发现，镉胁迫下巨大芽孢杆菌NCT-2的分泌蛋白是调控龙葵SA合成的重要诱导因子，但其种类和调控方式有待进一步深入研究。本课题拟采用分泌蛋白质组学技术系统解析镉胁迫下NCT-2菌株的差异分泌蛋白，结合生物信息学分析预测分泌蛋白种类，通过构建随机组合基因缺失突变菌株明确有效分泌蛋白；利用GST pull-down、EMSA等方法，考察分泌蛋白对SA合成酶及其转录因子的调控作用；通过有效分泌蛋白过表达、转染龙葵等手段促进SA合成，探讨微生物分泌蛋白提高龙葵萃取镉的普适性，揭示NCT-2菌株对龙葵SA合成的诱导机制。本研究为发挥芽孢杆菌等有益微生物的生物强化修复作用提供科学依据，同时为提高超积累植物萃取镉的效率提供理论依据和技术支持。

利用微生物强化植物萃取是修复土壤重金属污染的有效途径。本项目以有益微生物巨大芽孢杆菌NCT-2和镉超积累植物龙葵为研究对象，探讨了NCT-2菌株介导下龙葵水杨酸(SA)合成与细胞壁修饰对重金属镉的协同解毒机制，主要研究结果如下：（1）镉胁迫条件下，采用NCT-2菌株接种龙葵，可显著增强龙葵对镉的富集和转运，并提高其根部、叶片中的SA含量；（2）接种NCT-2菌株提高了龙葵SA合成途径的异构分支酸合成酶基因ICS1、调控SA积累的防卫蛋白基因EDS1、SA信号转录激活因子TGA1和谷胱甘肽S-转移酶基因GST的表达量，而病程相关蛋白基因PR1表达量显著下调；同时，NCT-2还通过激发龙葵体内金属抗性、转运或解毒功能蛋白基因的转录表达，提高了植物对镉的耐受和吸收积累能力；（3）NCT-2菌株主要通过胞内吸附镉并定殖于植物根内的方式提高了龙葵对镉的吸收富集，同时发现植物细胞内约50%的镉固定于细胞壁，并主要存在于细胞壁的果胶和半纤维素组分中，有益菌介导下龙葵通过重配镉在细胞壁不同组分中的比例、调整镉的不同化学形态等降低了镉对植物的生物毒性；（4）转录组学结合RT-PCR试验分析发现NCT-2介导下龙葵SA信号通路基因与细胞壁代谢相关基因的表达存在时间关联性；（5）采用微胶囊技术制备了NCT-2菌剂，并应用于强化超积累植物龙葵修复镉污染土壤，显著提高了植物对镉的萃取效率，为重金属污染土壤的植物萃取修复技术的实际应用奠定了基础。上述研究结果已发表SCI论文6篇，其中1篇入选全球前3%ESI高被引论文；申请发明专利2项。