三七自毒物质降解菌的特性和作用机制研究

Panax notoginseng is a valuable herbal medicine in China, but exists a serious problem of continuous cropping obstacles in the process of its growth. The accumulation of autotoxins is one of the causes of it. Our studies had shown that 1-G8, PEDBA41 and other strains obtained in previous studies had better abilities to degrade benzoic acid and saponins, all of which are the major autotoxins of Panax notoginseng. In this project, the activity of the isolated autotoxic degrading bacteria will be evaluated for benzoic acid and Panax notoginseng saponins, and the optimal strains will be selected which will apply to study its degradation characteristics. Meanwhile, the influences of degrading bacteria strains on soil microbial community will be studied after soil polluted with toxic substances, and then tracking their degradation process. .The metabonomic and transcriptome analysis of degradation will be carried out with LC-MS and other techniques, and the intermediates and key enzymes in the degradation pathway will be deduced, while verifying the activity of these enzymes. At the same time, the main products from the degradation of autotoxins will be separated and their effects on the growth of Panax notoginseng will be verified to explore the effectiveness of overcoming the obstacle of continuous cropping. The research results would be beneficial to the development of microbial inoculum, overcoming the continuous cropping barriers, and improving the yield and quality of Panax notoginseng.

三七是我国名贵中药材，连作障碍发生严重。自毒物质的积累是三七连作障碍发生的原因之一。课题组前期研究获得的1-G8、PEDB41等菌株具有强的降解三七自毒物质苯甲酸和总皂苷的能力。本项目在此基础上，从浓度效应、降解条件等方面对最优菌株进行降解特性研究；在外源添加自毒物质的土壤中加入降解菌，研究其对土壤微生物群落变化，并跟踪自毒物质的降解过程；运用代谢组学分析差异代谢物，结合转录组学推断降解途径中的关键基因，并进行基因表达分析；同时，分离自毒物质降解的主要转化产物，验证其对三七生长的影响以探讨克服连作障碍的有效性。研究结果对开发生物菌剂，缓解三七连作障碍、提高三七药材的产量和质量都具有重要的理论和实践意义。

三七是我国名贵中药材，连作障碍发生严重，制约了三七生产。连作土壤中自毒物质的降解对缓解三七连作障碍问题尤为重要。鉴于此，本项目以三七自毒物质降解菌为对象，历经4年，进行了降解菌对三七自毒物质的降解特性、降解机制、降解菌应用潜力探究3方面研究。主要工作及研究结果如下：.1.降解菌降解特性研究。利用单因素试验分别研究了皂苷降解菌SC3和苯甲酸降解菌SCB32的降解特性并优化降解条件，在以自毒物质为唯一碳源的液体无机盐培养基中，菌株SC3能在120h内将200mg/L三七总皂苷降解88.4%；能在96h内将400mg/L人参皂苷Rb1降解超过90%；在以苯甲酸为唯一碳源的液体无机盐培养基中，菌株SCB32能在48h内将800mg/L苯甲酸降解97.0%。.2.降解菌降解途径研究。分离并鉴定出菌株SC3降解人参皂苷Rb1产生的3个主要降解产物，并以此提出该研究中Rb1的降解途径为人参皂苷Rb1→绞股蓝皂苷XVII→人参皂苷F2→人参皂苷C-K途径；分离并鉴定出菌株SCB32降解苯甲酸产生的3个关键降解产物，提出SCB32菌株对苯甲酸降解是通过苯甲酸邻位降解途径。.3.降解机制研究。对SCB32菌株进行了全基因组测序，基因组大小为6.3Mbp，从基因组中共比对到55条苯甲酸降解基因及其同源基因，实时荧光定量PCR结果显示苯甲酸邻位降解途径上的基因在不同时间点有不同程度显著上调，该结果验证了菌株SCB32是通过邻位途径对苯甲酸进行降解。.4.研究证明了降解菌具有一定的应用潜力。SCB32及SC3降解自毒物质的产物不影响三七种子的活力；模拟土壤降解中，接种降解菌后苯甲酸的实际降解量是自然降解的10.5倍，Rb1实际降解量是自然降解量的3.2倍；盆栽试验中，接种降解菌后三七存活率最大提高至66.7%。