毒素在高粱靶斑病菌致病过程中的作用及其合成相关基因的表达

发布时间：2018-09-12 07:30

【摘要】：高粱靶斑病是由丝状真菌--平脐蠕孢菌(Bipolaris sorghicola)引起的一种高粱叶部真菌病害。该类病原菌能够产生二倍半萜类毒素--蛇胞菌素(ophiobolin),且该毒素来源于经典的MVA(甲羟戊酸)途径。本研究拟探讨蛇孢菌素在高粱靶斑病菌致病过程中的作用及不同条件下毒素合成关键酶编码基因的表达情况。首先在不同寄主表皮细胞中对比观察该病原菌的侵染特性,后采用MVA途径的抑制剂--洛伐他汀处理高粱靶斑病菌孢子悬浮液,将其接种于离体高粱叶片上观察病原菌致病性变化,并与创伤接种和人为添加毒素的病原菌致病性进行对比。后采用高效液相色谱-串联质谱法(LC-MS/MS)测定该病原菌在PDB液体培养基中所产毒素的种类和含量及洛伐他汀处理对其含量的影响;并结合蛇孢菌素处理后高粱叶片保护酶活性及质膜过氧化程度的变化,综合分析毒素在病原菌致病过程中的作用。同时,利用qRT-PCR技术对不同条件下该病原菌毒素合成关键酶编码基因(IPPI/FPPS/HMGR/GGPPS)的表达进行了检测。实验结果如下:1.高粱靶斑病菌可侵染多种植物。除了高粱外,单子叶植物(如大麦)和双子叶植物(如洋葱、拟南芥)均是该病原菌的侵染对象。其在四种植物表皮细胞中的侵染情况大致相同,均可从细胞表面和细胞间隙两个途径进入细胞内部。2.高粱靶斑病菌所产毒素以蛇孢菌素A为主。洛伐他汀处理可明显降低毒素含量,抑制高粱靶斑病菌菌丝在高粱叶表皮细胞中的侵染,导致病原菌的致病性显著下降。且在0-1500μg/ml浓度范围内,随着浓度的加大,病原菌致病性的抑制程度也随之增大,最佳处理时间和最佳处理浓度分别为24h和1000μg/ml。且人为添加蛇孢菌素和创伤接种均可恢复病原菌的致病性。此外该毒素可引起高粱叶组织细胞膜透性改变,膜脂过氧化加剧,MDA含量上升,且随着毒素处浓度的加大和处理时间的延长细胞膜透性和MDA含量持续增大。同时还可造成高粱叶片SOD酶活性下降,CAT、APX的酶活性升高。说明蛇孢菌素可影响高粱叶组织细胞膜透性,造成膜功能的紊乱,同时影响寄主保护酶活,使病原菌更易入侵植物。3.IPPI、FPPS、GGPPS和HGMR是高粱靶斑病菌毒素合成过程中的四个关键酶。我们通过Real Time PCR技术,检测了高粱靶斑病菌菌丝阶段这四个酶的编码基因(IPPI、FPPS、GGPPS、HGMR)在不同条件下的基因表达情况。结果显示:不同浓度洛伐他汀处理(25μM、50μM、100μM、200μM)均可导致毒素合成酶编码基因表达的下降,且随着处理浓度的加大抑制程度也随之增大。而相同浓度的洛伐他汀(100μM)在不同处理时间(2h、8h、24h、36h、48h)条件下,随着处理时间的推移,除了FPPS在8h和36h有反馈性提高外,高粱靶斑病菌菌丝阶段毒素合成酶编码基因IPPI、HMGR和GGPPS的表达在任一时间均明显下降。此外:我们发现不同温度、光照、PH条件下IPPI、FPPS、GGPPS表达模式相似,适当的高温(32℃)均可提高这三种基因的表达;HMGR的表达模式则与其相反,高温(32℃)反而不利于该基因的表达,PH4.0的弱酸环境则促进其表达,相比对照组升高近20倍,且HMGR基因对酸性环境非常敏感。
[Abstract]:Sorghum target spot disease is caused by filamentous fungus Bipolaris sorghicola. This pathogen can produce double sesquiterpene toxin, ophiobolin, and this toxin is derived from the classical MVA pathway. This study is to explore the pathogenesis of ophiobolin in Sorghum target spot pathogen. The infection characteristics of the pathogen were observed in different host epidermal cells. Lovastatin, an inhibitor of MVA pathway, was used to treat the spore suspension of sorghum target spot pathogen and inoculated on Sorghum leaves in vitro to observe the pathogenicity. The pathogenicity of the pathogenic bacteria was compared with that of wound inoculation and artificial addition of toxins. The toxins produced by the pathogen in PDB liquid medium were determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the effects of lovastatin treatment on the content of the toxins were also studied. At the same time, the expression of IPPI / FPPS / HMGR / GGPPS was detected by qRT-PCR under different conditions. The results were as follows: 1. Sorghum target spot pathogen can infect many plants. In addition to sorghum, monocotyledons (such as barley) and dicotyledons (such as onions, Arabidopsis) are infected by the pathogen. The infection of the pathogen in the epidermal cells of the four plants is similar, and it can enter the inner cells from the cell surface and intercellular space. 2. Lovastatin treatment can significantly reduce the toxin content, inhibit the infection of sorghum target spot pathogen hyphae in Sorghum leaf epidermal cells, resulting in a significant decrease in pathogenicity of pathogenic bacteria. The pathogenicity of pathogenic bacteria could be restored by adding anthropogenic serotonin and wound inoculation, respectively, 24 h and 1000 ug/ml. In addition, the toxin could cause membrane permeability change, membrane lipid peroxidation and MDA content increase in Sorghum leaf tissue, and cell membrane permeability and MDA content increased with the increase of toxin concentration and treatment time. The results showed that ophiocin could affect the membrane permeability of sorghum leaf tissue, cause membrane dysfunction, affect the activity of host protective enzymes, and make pathogens more likely to invade plants. 3. IPPI, FPPS, GGPPS and HGMR are the four key factors in the process of toxin synthesis of sorghum target spot pathogen. The gene expression of the four enzymes (IPPI, FPPS, GGPPS, HGMR) in the mycelial stage of Sorghum target spot pathogen was detected by Real Time PCR under different conditions. However, the expression of IPPI, HMGR and GGPPS in the mycelial phase of Sorghum target spot pathogen increased with the increase of the concentration of lovastatin (100 mu M) and the treatment time (2 h, 8 h, 24 h, 36 h, 48 h), except for the feedback of FPPS at 8 h and 36 h. In addition, we found that IPPI, FPPS and GGPPS expression patterns were similar at different temperatures, illumination and PH conditions, and the expression of these three genes could be increased at appropriate high temperatures (32 degrees Celsius); on the contrary, high temperatures (32 degrees Celsius) were not conducive to the expression of HMGR gene, and weak acid environment of PH4.0 promoted its expression. The control group increased by nearly 20 times, and the HMGR gene was very sensitive to acidic environment.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.14

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