新型外膜蛋白型β-1,6-葡聚糖酶GluM性质及其在珊瑚球菌EGB菌株捕食植物病原真菌中的作用研究

Cell wall lytic enzymes play important roles in the control of plant fungal pathogens. An outer membarane type β-1,6-glucanase GluM was isolated from myxobacterium Corallococcus sp. EGB in our previous research. GluM is the first reported outer membrane glycosidase, which can act act on cell wall of plant pathogenic fungi. GluM shows no similarity to any known families of glycosidases. We will biochemically characterize GluM and determine its dynamical parameters. Glycosidase domain of GluM will be clarified by partial deletion from its N terminal and expression of N terminal fragments of diffent length. Erro prone PCR will be employed to map the key residues related to the active sites of glycosidase domain. Antibody against GluM will be preprared and used to localize GluM in the cell membrane system and outer membrane vesicles by western blotting and immunogold labeling electron microscopy. GluM mutant strain of Corallococcus sp. EGB will be created by insertion mutation and expression enhanced strain will obtained by doubling its gene dose in the genome. Both strains will be used to test its predation capacity against Fursarium oxysporum. Mycelium of F. oxysporum treated with purified GluM will be checked by scanning and transmission electronic microscopy to examine its morphological changes. Changement in glucan composition of the treated mycelium will also be analyzed. The results will be used to clarify the mechanism of GluM against various plant fungal pathogens. gluM will be introduced to Arabidopsis thaliana to test its antifungal capacity against Botrytis cinerea. Transgenic Arabidopsis thaliana will be used to valuate the potential of GluM in fungal pathogen antagonism in dicotyledon.

细胞壁水解酶在植物抗真菌病害中发挥着重要作用。前期研究中从粘细菌珊瑚球菌EGB中分离到一个全新的外膜蛋白型β-1,6-葡聚糖酶GluM，是目前第一个被发现的外膜结合糖苷水解酶，该酶可以高效作用于病原真菌细胞壁。GluM与已报道的糖苷水解酶家族蛋白无任何同源性，本项目拟研究该蛋白的生化性质、动力学参数，通过截短表达、区段表达的方式确定其糖苷水解酶结构域，通过随机突变的方式考察其活性位点。利用脂质体及脂双分子层重建技术探讨其β-折叠桶结构域的可能功能。制备GluM的抗体，利用Western Blotting技术和免疫电镜技术确定GluM在EGB菌株中的定位。构建gluM缺失和过表达的EGB菌株，考察其对尖孢镰刀菌的捕食行为的影响，利用纯酶研究其作用于真菌细胞壁的机制，阐明GluM在EGB菌株捕食土传植物病原真菌的作用机理。在拟南芥中表达gluM基因，评估其在双子叶植物抗真菌中的应用潜力。

植物真菌病害严重威胁农作物的优质高产和人身健康，生产上急需高效绿色安全的生防手段。本项目基于研究基础中所获得一株具有广谱抗菌活性的粘细菌珊瑚球菌EGB及其分泌的β-1,6-葡聚糖水解酶，开展该水解酶的生物学功能和抗菌性质以及该蛋白在粘细菌捕食植物病原真菌中的作用研究。所获得的主要成果为：（1）确定了该水解酶为专一性切割β-1,6-糖苷键的中温糖苷水解酶，具有典型的β-折叠桶状结构，是已知唯一具有糖苷水解酶活性的外膜蛋白，其活性区域存在于N端350个氨基酸；（2）β-1,6-葡聚糖酶GluM除了部分存在于外膜，还存在于分泌的外膜囊泡中；（3）β-1,6-葡聚糖酶专一性的以植物病原真菌细胞壁中的β-1,6-葡聚为靶点，通过破坏细胞壁完整性抑制真菌的侵染；（4）酶处理尖孢镰刀菌黄瓜专化型菌株发现，真菌细胞壁损伤后引起真菌细胞内甘油积累，同时还检测到活性氧大量积累导致的细胞凋亡现象；（5）转基因植物抗病育种结果发现β-1,6-葡聚糖酶编码基因gluM赋予了拟南芥对灰霉病菌的抗性。基于以上结果，确定了外膜型β-1,6-葡聚糖水解酶为广谱抗真菌蛋白，直接参与粘细菌对植物病原真菌的捕食过程，该抗真菌蛋白具有自主知识产权，将在转基因抗病育种方面具有巨大的应用潜力，其产生菌株粘细菌珊瑚球菌EGB能够应用于植真菌病害的生物防治。本项目成果将为我国植物真菌病害的控制提供机制明确的抗性基因和生防菌资源。