根系分泌物介导特异微生物对连作太子参的致害机制研究

The rhizosphere biological process and underlying mechanism mediated by root exudates are highlighted recently and become a hot issue for revealing the detailed causes of medicinal plants continuous cropping obstacle. To understand the biological process and toxic mechanism on continuous cropping Radix Pseudostellariae, the protocol is designed based on our previous studies. Zherong Radix Pseudostellariae 2 as well as a specific Serratia marcescens isolated from its rhizosphere soil were used as experimental materials to study the ecological roles of Serratia marcescens in continuous cropping Radix Pseudostellariae. The Serratia marcescens labeled with green fluorescence protein would be constructed and incubated in the sterile soil and tissue culture media containg different concentrations of root exudates extracts, then the residual content of the extracts would be detected by GC-MS for identifying the detailed component which promote the strain growth, and the amounts of Serratia marcescens as well as the content of carbapenem in this test strain will also be measured using qRT-PCR in situ and HPLC. Furthermore, two engineered strains with deficiency and overexpression of swrI from quorum sensing system in Serratia marcescens would be constructed and assessed the effects of quorum sensing on Radix Pseudostellariae tissue cultured seedling. Based on the researches mentioned above, the effects of Serratia marcescens, its fermentation supernatant liquid and quorum sensing signal molecules on Radix Pseudostellariae tissue cultured seedling would be bioassayed, and the differential proteins would be analyzed using the differential proteomic approach. It is expected to uncover the rhizospheric biological process and its toxic mechanism of specific Serratia marcescens on continuous cropping Radix Pseudostellariae.

根系分泌物介导下的根际生物学过程与机制是揭示由连作引起的药用植物栽培障碍效应的重要环节，已成为当前植物连作障碍研究的重要内容和热点问题。本研究以"柘荣太子参2号"为材料，并利用从连作太子参根际分离与鉴定得到的一种特异粘质沙雷氏菌，研究该菌响应太子参根系分泌物介导连作障碍效应的根际生物学过程与机制。试验拟转绿色荧光蛋白基因入该菌，接种至添加不同浓度根系分泌物的无菌土壤和组培基质中，分析其残留量，确认何种组分对该菌呈促进作用，并原位和定量检测该菌数量和Carbapenem含量；进一步敲除和过表达该菌群体效应swrI基因，接种至太子参组培苗基质中，确认群体效应是否介导该菌对太子参产生致害作用；并在含有根系分泌物的培养基中发酵该菌，分别评价菌株、发酵液及群体效应信号分子对太子参生长的影响，分析其差异蛋白表达情况，阐明互作机制

项目背景：根系分泌物介导下的根际生物学过程与机制是揭示由连作引起的药用植物栽培障碍效应的重要环节，已成为当前植物连作障碍研究的重要内容和热点问题。.主要研究内容：.1、分析太子参连作条件下土壤群体感应产生菌和淬灭菌的种类和数量及对太子参苗生长的影响；.2、验证群体感应系统调控根际特异粘质沙雷氏菌致害太子参；.3、确认群体感应系统通过调控特异粘质沙雷氏菌抗菌物质合成、胞外酶分泌、浮游和挥发性物质合成来实现对太子参的侵染与致害；.4、使用绿色荧光蛋白标记特异粘质沙雷氏菌，考察特异粘质沙雷氏菌在太子参根际土壤响应太子参根系分泌物增殖情况以及趋化行为；.5、分析太子参根系分泌物主要成分，考察太子参根系分泌物和块根提取物对特异粘质沙雷氏菌生长的影响；.6、通过转录组测序分析特异粘质沙雷氏菌响应根系分泌物致害太子参过程中转录组差异。.重要结果与关键数据：.1、未种植土壤、正茬和重茬土壤中群体感应产生菌分别占可培养微生物的5%、12%和15%，呈现增长趋势，而群体感应淬灭菌仅在正茬土壤中获得1株；.2、群体感应产生菌均为特异粘质沙雷氏菌，属于SwrI-SwrR型群体感应系统，群体感应淬灭菌为苏云金芽孢杆菌，可分泌信号分子降解酶aiiA，实现对群体感应系统的淬灭；.3、群体感应产生菌粘质沙雷氏菌可明显致害太子参，导致烂根和叶片枯萎，而群体感应淬灭菌可有效缓解特异粘质沙雷氏菌对太子参的致害；.4、特异粘质沙雷氏菌致害太子参苗受群体感应系统调控，表型验证表明群体感应通过调控粘质沙雷氏菌抗菌物质的合成、胞外酶的分泌、浮游和挥发性物质的合成实现对太子参的致害和侵染；.5、太子参根系分泌物和块根提取物可明显促进特异粘质沙雷氏菌的增殖，进而成为优势菌。.6、目前已发表SCI论文6篇，中文论文4篇，培养研究生4人。.科学意义：根系分泌物介导下的根际生物学过程与机制是揭示由连作引起的药用植物栽培障碍效应的重要环节，本研究围绕太子参根际群体感应产生菌，较为完整的研究了太子参根系分泌物对群体感应产生菌生长的影响、群体感应产生菌致害太子参的机制以及太子参如何响应群体感应产生菌的致害，获得的数据对于深化理解太子参连作障碍的化学生物学机理有着较为重要的理论价值和实际应用价值。