人参-病原菌互作的分子生态机制研究

Ginseng is a highly valued herb which has a large number of structurally different ginsenosides present in relatively high abundance, while it is attacked in commercial ginseng gardens by a diverse array of fungi including Cylindrocarpon destructans. The ecological mechanism of plant-pathogen interactions are not clear. Our previous study found that ginsenosides from panax ginseng C.A Maye inhibited the growth of the five ginseng nonpathogens, while it promoted the growth of the ginseng pathogen C. destructans. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can hydrolyze ginsenosides by extracellular glycosidase.These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi C. destructans might counter their toxicity by converting ginsenosides into growth or host recognition factors using ginsenoside-degrading enzyme. In order to clarify the significance of the ginsenoside-degrading enzymes in the pathogenic behavior of C. destructans and the ecological role of ginsenosides in ginseng-pathogen interactions, we intend to study the purification and biochemical characterization of these saponinases, and then study the gene cloning and characterization of them with the peptide information provided above, furter perform targeted disruption of the genes, assay the sensitivity of knockout mutants to ginsenosides and the pathotypic behavior of mutants on ginseng. Our findings may provide more evidences for the law of plant-microbe interactions and the theory of biological co-evolution. In addition, it will provide ideas for the effective control of ginseng diseases.

名贵药用植物人参的真菌病害十分严重，而人参与病原真菌相互作用的分子生态机制尚不明确。我们前期研究表明：人参可能通过次生代谢物人参皂苷防御病原菌的侵害，而人参最主要的病原菌毁灭柱孢菌可能是通过产生人参皂苷降解酶解除了人参皂苷毒性,并且将降解产物转化为自身的生长促进因子或是寄主识别因子，从而寄生成功。为了验证上述假设，本项目拟研究毁灭柱孢菌产生的人参皂苷降解酶的分子特征和酶学性质，在此基础上分离鉴定该酶基因的结构和特征，进而用基因敲除方法构建人参皂苷降解酶基因缺失突变体，研究突变体对人参皂苷的敏感性和对人参的致病力，从分子水平上探讨毁灭柱孢菌人参皂苷降解酶在其致病性中的作用，阐明人参皂苷在人参-病原菌互作中的生态功能及其分子机制。研究结果将为阐明植物-微生物相互作用的规律和生物共进化原理提供证据，也将为人参病害防治提供可行思路。

名贵药用植物人参的真菌病害十分严重，而人参与病原真菌相互作用的分子机制尚不明确。前期研究表明：人参可能通过次生代谢物人参皂苷防御病原菌的侵害，而人参最主要的病原菌毁灭柱孢菌可能是通过产生人参皂苷降解酶解除了人参皂苷毒性,并且将降解产物转化为自身的生长促进因子或是寄主识别因子，从而寄生成功。为了验证上述假设，本项目测定了毁灭柱孢菌和四种非人参病原菌对人参皂苷单体Rb1、Rb2、Rc、Rd、Rg1和Re的敏感性，调查了毁灭柱孢菌对人参皂苷的酶促降解与其致病性的关系，研究了人参皂苷对毁灭柱孢菌细胞膜的作用、人参皂苷降解酶的分子特征和酶学性质以及毁灭柱孢菌基因组结构及其致病机制。结果表明，六种人参皂苷单体对四种非人参病原真菌均显示了生长抑制作用，而对毁灭柱孢菌显示了不同的活性。二醇型人参皂苷单体Rb1和Rb2显著刺激了人参锈腐病菌的生长，二醇型人参皂苷的主要酶解产物G-XVII和F2对毁灭柱孢菌和四种非人参病原菌均有很强的抑制活性，人参皂苷酶解产生的单糖不是促进人参锈腐病菌生长的碳源，四种非人参病原菌也能够产生人参皂苷水解酶。该结果说明人参皂苷是化学防御物质，毁灭柱孢菌对人参皂苷的酶解不是其致病机制。人参皂苷Rb1具有破坏真菌细胞膜的作用，说明其促进毁灭柱孢菌菌丝生长的机制不是通过人参皂苷与细胞膜的信息交流实现的。毁灭柱孢菌产生的一种人参皂苷降解酶，水解Rb1为中间产物G-XVII，终产物F2，该酶相对分子量35KD左右，不是单纯蛋白，可能有辅酶或辅基参与，也可能是复合蛋白。本研究提取毁灭柱孢菌基因组，用Illumina HiSeq2500通过PE125策略测序，用SOAP de novo进行组装，得到一个60.4 Mb大小的基因组草图，GC含量为51.8%，编码10031个基因，有490个基因为糖类化合物相关酶类（CAZymes），E-value小于1e-5，包含264个糖苷水解酶（GH）。本项目首次研究人参皂苷单体及其酶解产物的抗真菌活性以及酶解机制与毁灭柱孢菌致病性的关系，研究结果为阐明人参-病原菌相互作用的机制提供了证据，为人参病害生物防治提供了可行思路。