【作者】 乔广行；

【导师】 王克荣；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 栗疫病菌是栗树上毁灭性的病害之一,其引起果树枝干溃疡病是栗生产上的病害之一。由于栗疫病菌难以防治,普通的化学防治比较困难。栗疫病菌的细胞死亡与相关基因的研究,可以为寻找新的杀菌剂靶标,设计新的病害控制策略提供理论指导。营养体不亲和性诱导栗疫病菌菌株间程序性细胞死亡为了验证营养体不亲和性导致细胞凋亡的特征,主要进行了融合细胞基因组DNA降解的琼脂糖电泳检测,细胞学研究主要是细胞核变化的染色,活性氧产生的测定,脂肪粒染色以及融合细胞的死活检测。实验结果表明混合孢子涂板基因组DNA降解琼脂糖凝胶电泳检测DNA梯状条带,这一结果为营养体不亲和性诱导的细胞死亡提供了新的证据,细胞学研究观察到细胞核的降解,活性氧的变化以及脂肪粒的积累,融合细胞的死亡,与凋亡类细胞死亡相似。外源过氧化氢对栗疫病菌低毒力菌株EP713四种抗氧化酶的影响用外源的氧化压力模拟寄主的氧迸发影响,用10 mM过氧化氢处理低毒力菌株EP713与野生型菌株EP155不同时间取样,测定处理菌丝的四种抗氧化酶活性,结果表明EP155与EP713的四种抗氧化酶的活性达最大值时的活性有差异,但谷胱甘肽还原酶与谷胱甘肽过氧化物酶出现最大值的时间野生型菌株比低毒力菌株较晚,活性较高。说明低毒力病毒对栗疫病菌抗氧化能力可能有影响。过氧化氢诱导栗疫病菌分生孢子细胞死亡对外源氧化压力诱导栗疫病菌分生孢子的细胞死亡进行了研究。过氧化氢亚致死与致死浓度为10 mM与150 mM；对过氧化氢影响分生孢子萌发的时间效应进行了分析和细胞学研究,细胞学主要观察细胞核的变化,活性氧与一氧化氮的产生,以及电泳检测DNA降解,实验结果表明外源的氧化压力引起细胞核的变化,活性氧随处理时间增长不断增多,一氧化氮产生主要是150 mM处理30 min,电泳结果没有检测到DNA梯状条带,表明外源的氧化压力诱导栗疫病菌分生孢子的细胞死亡,这种细胞死亡细胞核发生降解,且这种细胞死亡与活性氧和一氧化氮的产生相关。栗疫病菌核糖基化因子1(ARFl)基因功能的表型效应在COGEME数据库栗疫病菌EST中搜索到CpARFl,克隆了CpARFl全长,并进行了聚类分析与同源性比较,该基因具有保守的高尔基体结合的结构域MXXE;利用转录基因沉默技术研究了该基因在栗疫病菌中的功能,沉默突变体表现出对硫酸铜与氟化钠的敏感,漆酶产生减少,无性孢子产生减少或不产生,并且导致致病性减弱。结果表明栗疫病菌CpARF1可能影响栗疫病菌对硫酸铜与氟化钠的敏感性,漆酶产生,无性繁殖以及致病性。栗疫病菌泛素核糖体L40融合蛋白基因功能的表型效应在COGEME数据库栗疫病菌EST中搜索到CpUFP,克隆并分析了栗疫病菌中泛素融合蛋白CpUFP。该基因氨基酸含有典型的结合泛素结合酶的结构域,系统进化上与其它丝状子囊菌的亲缘关系较近。利用转录基因沉默技术研究了该基因在栗疫病菌中的功能,沉默突变体表现出对过氧化氢与氯化钠的敏感,漆酶的产生减少,无性孢子产生减少或不产生,并且导致致病性减弱。结果表明CpUFP基因可能影响栗疫病菌对过氧化氢与氯化钠的敏感性、漆酶的产生、无性繁殖以及致病性。更多还原

【Abstract】 Chestnut blight fungus (Cryphonectria parasitica) is very destructive disease of chestnut tree; it causes the chestnut tree stem canker and one of the diseases of chestnut production. Because of the difficulty of control this disease; the chemical control of this disease is difficult. Study on the chestnut blight fungal cell death and related genes fuction, it is possible to find the new target of chestnut blight control and design the new disease control strategy to give the new theory of direction.Vegetative incompatibility induced programmed cell death between strains of chestnut blight fungal fusion cell The study of vegetative incompatibility induded cell death of chestnut blight fungus was done. In order to test the hypothesis whether vegetative incompatibility results in nuclear degradation, a characteristic of apoptosis, the agar electrophoresis was mainly used to detect the degeneration of fusion cell genome DNA of chestnut blight fungus, the cytological observation on the change of the nuclear in hyphae between incompatible strains, the production of the reactive oxygen species, the accumulation of lipid droplet and the identification of the live and dead cell. The results showed that the electrophoresis detected the DNA ladder, which provided the new evidence of vegetative incompatible cell death. Hyphal fusion cells between vegetative incompatible strains were compartmentalized by septal plugging and contained heavily degraded nuclear DNA. Reactive oxygen species accumulation decreased in hyphal fusion cells, the accumulation of lipid droplets increased and finally the hyphal fusion cell died. Morphological features of hyphal compartmentation and death during vegetative incompatibility can be described as a form of programmed cell death.Effect of exogenous hydrogen dioxide on four antioxidases of Chestnut Blight hypovirulent strain EP713 When plant pathology infected host plant, host plant produced oxide burst and protected itself from the fungi attacking. The exogenous oxide stress was used to mimic the influence of the host oxide burst, detected the change of antioxidase activity. The 10 mM H2O2 was used to treat the strain of EP155 and EP713 and the samples were taken at different treatment time. The proteins were extracted and detected the antioxide enzymes activity including the activity of catalase (CAT), superoxide dismutase (SOD) Glutathione peroxidase (GPx) and Glutathione reductase (GR). The results showed that the maximum activity of four kinds of antioxidases is different between EP155 and EP713, however, the time of the GR and GPx reaching maximum activity EP155 is later than EP713, and the activity of the four kinds of enzyme of EP155 is higher than EP713. These results showed that the activity and the time of four antioxdase to reach maximum activity are different.Hydrogen dioxide induced the cell death of chestnut blight conidia Chestnut blight fungus (C. parasitica) is one of the major diseases of chestnut tree worldwide. It is mainly spread by the conidia of this fungus. We were interested in the conidia response to the exogenous oxide stress. Sublethal and lethal dose of hydrogen dioxide was 10 mM and 150 mM; the germination rate of the conidia was analyzed and the cytology research was done along with the different treatment time. The cytology was mainly detected:the change of nuclear, reactive oxygen species and nitric oxide generation. The agar electrophoresis was detected the degeneration of genome DNA. The results have been shown that the change of the nuclear, the ROS generation increasing along with the treatment time, nitric oxide only appeared at the treated time of 30 min in 150 mM. The results of this experiment have been shown exogenous oxide stress induced the conidia of chestnut blight cell death, which is associated with the degeneration of nuclear and the generation of ROS and NO.Functional effects of ADP-ribosylation factor 1 from C. parasitica on its phenotypes ADP-ribosylation factor 1 gene CpARFl in C. parasitica was cloned and analyzed. The amino acid residues of this gene have conserved Golgi binding domain MXXE, the phylogenetic evolution is near to other ascomycytes. Using the gene silencing technique,99 transformants were attained,3 of these transformants were silenced mutants. The phenotypes of silenced mutants were detected, which were included in the sensitivity to CUSO4 and NaF, the accumulation of laccase, the reduction of conidia production and the decrease of pathogenicity. The results showed that CpARFl has influence on the sensitivity to CUSO4 and NaF, the production of laccase, the asexual reproduce and the pathogenicity.Functional effects of ubiquitin and ribosome L40 fusion protein from C. parasitica on its phenotypes Ubiquitin fusion protein (UFP) gene CpUFP in C. parasitica was cloned and analyzed. The amino acid residues of this gene have conserved ubiquitin conjugation enzyme binding domain, the phylogenetic evolution is near to other ascomycytes. Using the gene silencing technique,63 transformants were attained,2 of these transformants were silenced mutants. The phenotypes of silenced mutants were detected, which were included in the sensitivity to H2O2 and NaCl, the accumulation of laccase, the reduction of conidia production and the decrease of pathogenicity. The results showed that CpUFP has influence on the sensitivity to H2O2 and NaCl, the production of laccase, the asexual reproduce and the pathogenicity.更多还原