促进植物富硒的弧形柄杆菌T5M6氧化硒的机制与根土定植规律

Selenium is an essential trace element for life. Selenium deficiency of human and animal causes cardiomyopathy and other 40 kinds of diseases. One billion of the population are in the Se-starving status in the world. The root mainly absorbs Se(VI) and then convert it to organic selenium entering the food chain. Soil microorganism is involved in the biogeochemical cycle of the selenium. The microbial oxidation of selenium is the premise of plant absorption and transformation of selenium, but it remains unknow. The Caulobacter vibrioides T5M6 was isolated from rhizosphere in Se-rich soil. T5M6 had the ability to oxidize Se(0) to Se(VI) and to promote plant absorption of selenium. Based on the completed genome sequencing of T5M6, this study would focus on 1) elucidating the metabolic pathway of Se oxidation by using the comparison of proteomics, combined with the analysis of transcription of up/down expressed proteins; 2) cloning the key genes of Se oxidation by using proteomic results and transposon insertional mutagenesis; 3) monitoring the colonization of T5M6 in bulk soil and rhizosphere by using GFP fluorescence gene marker in T5M6 under elemental Se in the pot culture. Meanwhile, the bacterial community structure will be analyzed by high-throughput sequencing. In addition, the correlation between T5M6 colonization, Se oxidation and Se uptake in plant would be investigated. These investigations will be a comprehensive interpretation of microbial transformation of Se in soil, in particularly the relationship between Se oxidation and plant uptake of selenium, as well as serve as human and animal health in Se-deficient area via promotion of Se uptake in plant.

硒是生命必需的微量元素，人畜硒摄入不足会导致心肌病等多种疾病，全球因食物链导致的严重缺硒人口约十亿。植物根主要吸收硒酸盐再转化为有机硒进入食物链；土壤微生物是硒循环的主要推动者，其中微生物氧化硒是植物吸收转化硒的前提，但相关研究匮乏。弧形柄杆菌T5M6 等分离自富硒土壤根际，可将单质硒氧化成硒酸盐，并促进植物吸收硒。本研究在已完成的全基因组测序基础上，利用比较蛋白质组，结合差异蛋白转录水平分析，解析硒氧化的相关代谢路径；利用组学结果和转座插入突变技术，克隆硒氧化关键基因，明确硒氧化物质；在盆栽中添加单质硒，以荧光基因标记T5M6，定量追踪其在土壤和根际的定植情况，同时采用高通量测序分析土壤和根际细菌群落结构，阐释T5M6定植、硒氧化和植物吸收硒的关联性。通过上述研究，将为全面解读微生物转化土壤硒，特别是硒氧化和植物吸收硒的关系提供基础数据，为促进广大缺硒地区植物富硒从而维护人畜健康服务。

植物主要吸收土壤中氧化态的硒而进入食物链以供人体和动物吸收并发挥功能，但微生物介导的硒的氧化所知甚少。本课题基于柄杆菌T5M6、农杆菌T3F4等细菌，解析硒氧化与植物富硒之间的关联性；发掘参与单质硒Se(0)到Se(IV)的关键氧化酶（基因）。具体结果如下。1）首次证实多种细菌可将硒代蛋氨酸和硒代胱氨酸等有机硒氧化成Se(IV)。土壤中氧化有机硒的细菌类群丰富，除农杆菌T3F4外，新分离获得共计20属33株细菌。将其中4株菌分别加入到富硒土壤中，发现其具备氧化和溶解硒的作用，显著增加土壤中可利用硒的含量。2）利用GFP荧光标记农杆菌T3F4，接种到小白菜盆栽土中，21天时活菌计数表明该菌在土壤中和小白菜体内稳定定殖。在添加5 mg/kg Se(0)或Se(IV)的土壤中，T3F4均显著促进小白菜对土壤硒的吸收。进一步测定土壤硒形态，表明T3F4通过氧化和溶解作用增加可溶性硒的含量，进而促进植物富硒。柄杆菌T5M6具有同样的机制。3）通过差异蛋白质组、基因敲除与互补、体外酶活等实验，证明农杆菌T3F4中NAD(P)H依赖型黄素氧化还原酶(YrpB)参与了Se(0)到Se(IV)的氧化。总之，首次发现细菌可将硒代蛋氨酸等有机硒氧化成Se(IV)，揭示土壤中存在丰富的有机硒氧化细菌类群，获得了硒生物地球化学循环的新认知；阐释了硒氧化细菌通过氧化和解硒作用，提高土壤可利用硒的含量，进而促进植物吸收硒，对植物硒强化生产实践具有指导意义；发现了农杆菌T3F4氧化单质硒的关键基因yrpB，对硒的生物地球化学循环研究具有支持作用。