摘要
【目的】以枣黑斑病菌产生的细胞壁降解酶为研究对象,明确其产生细胞壁降解酶种类和活性,探讨细胞壁降解酶在病菌致病中的作用,为细胞壁降解酶参与病原菌侵染机理的研究提供理论依据。【方法】利用3,5-二硝基水杨酸(DNS)法和考马斯亮蓝(Bradford)法,通过紫外-可见分光光度计测定反应混合物吸光度,根据酶反应所释放的还原糖计算细胞壁降解酶活性。【结果】枣黑斑病菌在寄主体外不同碳源诱导下均能产生多聚半乳糖醛酸酶(Polygalacturonase,PG)、羧甲基纤维素酶(Carboxymethyl cellulase, Cx)、β-葡萄糖苷酶(β-glucosidase)、木聚糖酶(Xylanase)、聚甲基半乳糖醛酸酶(Polymethylgalacturonase, PMG)和果胶甲基反式消除酶(Pectin methyltranseliminase, PMTE)等6 种细胞壁降解酶(Cell wall degrading enzyme,CWDE),但细胞壁降解酶活性存在一定差异,以骏枣果肉为外源诱导物,枣黑斑病菌产生β-葡萄糖苷酶活性明显高于其他5 种酶,说明β-葡萄糖苷酶在枣黑斑病菌致病过程中有着重要的作用,而以滤纸为诱导物产生的β-葡萄糖苷酶活性高于其他诱导物,其活性高达10.104 U·mg-1。枣黑斑病菌侵染枣果后产生的细胞壁降解酶种类及活性与体外不同碳源诱导的结果一致,可产生6 种细胞壁降解酶,其中β-葡萄糖苷酶活性高于其他5 种酶,且病健交界处β-葡萄糖苷酶的活性明显高于受侵染后的发病部位和未被侵染的部位。【结论】枣黑斑病菌在致病过程中起关键作用的细胞壁降解酶为β-葡萄糖苷酶,且在侵染过程中病健交界处的活性最高,而在寄主体外诱导β-葡萄糖苷酶最佳碳源为滤纸诱导物。
【Objective】The cell wall degrading enzymes produced by the black spot pathogen were studied, and their activities were determined. The role of cell wall degrading enzymes in the pathogenesis of the pathogens was discussed.【Methods】Jujube black spot disease was isolated from jujube in southern Xinjiang. Pathogen hyphae were separated from PDA culture medium. The hyphea were added into induction media containing sucrose, pectin, cellulose, filter paper powder, cotton powder, or jujube pulp as the inducers. The media were shaken in a shaker incubator set at 25 ℃ for 7 days, vacuum filtered to remove hyphae and spores, and centrifuged at 4 ℃ for 30 minutes at 10 000 r·min^-1. The supernatant was used as the crude enzyme solution. The crude enzyme solution was vacuum filtered, and ethylenediamine tetraacetic acid was added to the filtrate. Then ammonium sulfate was added slowly with gentle stirring until 60% saturation. The mixture was allowed to stand at 4 ℃ for 5 hours and centrifuged at 4 ℃ 15 000 r·min^-1 for 20 minutes. The supernatant was discarded, and the precipitation was dissolved with acetic acid-sodium acetate buffer and placed in a dialysis bag. In the same buffer, dialysis was carried out at 4 ℃ for 48 hours and the dialysate was changed every 12 hours. The purified enzyme was used for enzyme activity determination. Crude enzymes were also extracted in vivo from different parts of jujube diseased fruit inoculated with spore suspension. The tissues were cut into slices and put into mortar, grinded with sodium chloride at 4 ℃, centrifuged at 4 ℃ and 5 000 r · min-1 after filtration, and the supernatant was collected as the crude enzyme for later use. Activities of six cell wall degrading enzymes were measured using the 3, 5-dinitrosalicylic acid (DNS) method and the Coomassie blue staining method. Among them, the activities of polygalacturonase, carboxymethylcellulase,β-glucosidase, xylanase and polygalacturonase were measured by the 3, 5- dinitrosalicylic acid method using pectin, carboxymethyl cellulose, salicin and xylan as substrate, respectively. Substrate solution was prepared with citric acid buffer. Using citric acid buffer solution as the blank, the enzymic hydrolysis was carried out at 50 ℃ for 30 to 60 minutes. The absorbance of the reaction mixture was measured by UV-Vis spectrophotometer, and the the enzyme activities were calculated according to the reducing sugar released. The pectin methyl trans-eliminating enzyme was determined by Coomassie Brilliant Blue method. The color density of the protein-Coomassie brilliant blue complex solution was proportional to protein concentration. The differences in color of the solution created by enzymic reactions was used to calculate the cell wall degrading enzyme activity.【Results】Jujube black spot bacteria produced six cell wall degrading enzymes under the induction of different carbon sources. They were polygalacturonase, carboxymethylcellulase,β-glucosidase, xylanase, polymethylgalacturonase and pectin methyl transeliminase. However, there were some differences in activity among enzymes under different inducers. When the jujube pulp was used as the inducer, the activity of β-glucosidase produced by the black spot pathogen was significantly higher than that of the other five enzymes, indicating that β- glucosidase played an important role in the pathogenesis of black spot pathogen. Filter paper as the inducer also produced the highest β-glucosidase activity. The activities of cell wall degrading enzymes produced by jujube black spot in infected jujube fruit was consistent with the results induced by different carbon sources in vitro, with the activity of β-glucosidase being higher than the other five enzymes. Moreover, the activity of β-glucosidase at the junction of diseased and healthy parts was the highest among different tissues sites.【Conclusion】Different inducers have different effects on the cell wall degrading enzymes secreted by the black spot pathogen, and the inducer most favorable for pathogen growth has an important effect on the enzyme production. Filter paper can be used as a good inducer of β-glucosidase. Combining the enzyme activity assay results in vitro and in vivo, it is speculated that β-glucosidase is the major cell wall degrading enzymes that play a role in the pathogenesis of the black spot pathogen. The key part of the impact is at the edge of the diseased tissue.
作者
王鹏程
郝海婷
王兰
凌新慧
WANG Pengcheng;HAO Haiting;WANG Lan;LING Xinhui(Scientific Observing and Experimental Station of Crop Pests in Alar, Ministry of Agriculture/Southern Xinjiang Key Laboratory of IPM of Tarim University/Engineering Laboratory of Featured Fruit Tree in Southern Xinjiang, Tarim University, Alar 843300,Xinjiang, China)
出处
《果树学报》
CAS
CSCD
北大核心
2019年第7期903-910,共8页
Journal of Fruit Science
基金
国家自然科学基金(31660504)
兵团红枣新品种产业科技特派员创新创业示范项目(2016GA003)
关键词
枣
黑斑病菌
细胞壁降解酶
活性
致病性
Jujube
Black spot pathogen
Cell wall degrading enzyme
Activity
Pathopoiesis
