【作者】 吴方丽；

【导师】 曲东； 张兴；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2008， 博士

【摘要】 丙烷脒为烷基脒类化合物,其医药活性临床应用始于20世纪40年代,由于其良好的治疗作用至今临床仍有应用。20世纪90年代发现了丙烷脒对灰霉病菌(Botrytis Cirerea)的农用抑菌活性,研究表明丙烷脒为内吸性杀菌剂,可被植物体吸收、输导和代谢,具有保护和治疗双重功效,主要用于防治田间和大棚内蔬菜、果树和经济作物上由灰霉病菌引起的多种植物病害。毒性试验和环境生态试验表明,丙烷脒是一种低毒的化合物,其急性经口、经皮毒性均属于低毒,对皮肤、眼睛无刺激作用,对皮肤无致敏作用,无致畸、致突变作用。虽然丙烷脒对灰霉病菌等相关的病原菌具有良好的抑菌效果,但对其抑菌机理却是知之甚少。因此,本论文以番茄灰霉病菌为供试病原菌,主要从细胞生物学、生物化学和分子生物学等方面入手,对丙烷脒的抑菌机理进行较为系统的研究。取得如下结果:1、丙烷脒对番茄灰霉病菌菌丝形态和超微结构的影响(1)采用扫描电镜观察发现,随着丙烷脒处理时间的延长对番茄灰霉病菌菌丝的生长形态产生如下影响:A、菌丝塌陷现象逐渐严重;B、生长点出现分枝增多和缢缩;C、内含物外渗现象逐渐明显。(2)透射电镜观察发现,丙烷脒处理后番茄灰霉病菌超微结构发生明显的变异,其主要表现为:A、线粒体异常增多,表现出基质型肿胀现象;B、线粒体液泡化,最终形成大的液泡;C、病菌细胞内含物外渗,细胞出现质壁分离和空腔细胞;D、线粒体囊泡化解体和细胞组织崩解,细胞趋于死亡等现象。2、丙烷脒对番茄灰霉病菌能量代谢的影响(1)用电导率法测定了不同浓度丙烷脒对番茄灰霉病菌电导率的影响,试验结果表明:番茄灰霉病菌细胞膜通透性增加,与电镜观察到的番茄灰霉病菌细胞内含物外溢和细胞质壁分离等试验现象相吻合。表明番茄灰霉病菌细胞膜的结构完整性受到破坏。(2)用不同浓度的丙烷脒处理番茄灰霉病菌菌丝和孢子,进而测定其呼吸速率、线粒体呼吸功能和氧化磷酸化效率。试验结果表明,随着丙烷脒处理浓度的增加,菌丝、孢子的呼吸速率逐步下降;番茄灰霉病菌Ⅲ态呼吸速率、Ⅳ态呼吸速率和呼吸控制率也呈下降趋势;磷氧比和氧化磷酸化效率均呈下降趋势。表明丙烷脒处理抑制了番茄灰霉病菌的呼吸作用及能量代谢。3、丙烷脒对番茄灰霉病菌物质代谢的影响用不同浓度的丙烷脒处理番茄灰霉病菌,然后检测番茄灰霉病菌体内的总糖、还原糖、蛋白质及脂肪的含量变化。试验结果表明:A、随着丙烷脒处理浓度的升高,番茄灰霉病菌体内的总糖、还原糖和蛋白质的含量呈先升高后下降的趋势;而脂肪的含量则一直上升;B、番茄灰霉病菌体内的总糖、还原糖、蛋白质和脂肪的含量与未受丙烷脒处理的番茄灰霉病菌体内的总糖、还原糖、蛋白质和脂肪的含量相比均显著升高。表明番茄灰霉病菌体内的分解代谢受到抑制。4、丙烷脒对番茄灰霉病菌线粒体复合酶及ATP酶活性的影响(1)丙烷脒对离体番茄灰霉病菌线粒体复合酶及ATP酶活性的影响。用不同浓度的丙烷脒处理提取后的番茄灰霉病菌线粒体复合酶及ATP酶后,立即测定酶的活性,发现与未受丙烷脒处理的番茄灰霉病菌线粒体复合酶及ATP酶活性基本相同,表明丙烷脒抑制能量代谢并不是通过与番茄灰霉病菌线粒体复合酶及ATP酶相互结合而发生作用的。(2)丙烷脒对活体番茄灰霉病菌线粒体复合酶及ATP酶活性的影响。先用不同浓度的丙烷脒处理番茄灰霉病菌,再提取其线粒体复合酶及ATP酶进行活性测定。试验结果表明:当用不同浓度的丙烷脒处理番茄灰霉病菌1 d后,线粒体复合酶Ⅰ、Ⅱ、Ⅳ及ATP酶的活性受丙烷脒作用后逐步升高,而线粒体复合酶Ⅲ的活性却明显下降。而用不同浓度的丙烷脒处理番茄灰霉病菌3 d后,线粒体复合酶Ⅰ、Ⅱ、Ⅲ、Ⅳ及ATP酶的活性都明显下降。综上所述,结合芳香族脒类化合物在医学中的作用机理,初步推断丙烷脒对番茄灰霉病菌的抑菌机理是:通过与编码线粒体复合酶Ⅲ中的某个亚基的基因结合,从而导致该基因的表达受到抑制,进而致使整个线粒体复合酶Ⅲ的活性下降,呼吸过程中的电子传递受阻,机体的能量合成受到抑制,导致机体的能量供应不足,最终表现出番茄灰霉病菌的线粒体增多,细胞膜破裂、生长发育受到抑制,菌丝和细胞形态严重变形,直到死亡。更多还原

【Abstract】 Aromatic diamidines, and in particular bis(amidinophenoxy)-alkanes, have 70 years history in the treatment of human diseases and show diverse pharmacological activity. Propamidine is an antimicrobial agent used clinically for the treatment of Acanthamoeba keratitis and other corneal infections. But limited oral bioavailability and both acute and chronic toxicity have slowed down the studies on development of this class of compounds. The antifungal activity of pentamidine against Botrytis cinerea was found incidentally by NZYM Inc. in 1990s, a large number of analogues have been synthesized and propamidine have been used in the field for treating crop’s disease. Propamidine is a new type, low toxicity, high efficiency and without side effects of pollution antiseptic which has the particular protective and therapeutic action to many plants pathogeny. It is used to prevention and cure many field, vegetable of shed, fruit tree and economical crop’s disease which caused by Botrytis cinerea Pers. Propamidine has the diplex protective and therapeutic action which can absorb, distribute and metabolize in the plant.Although propamidine has strong antifungal activity and has been used to treat crop’s disease, few or no literatures reported the inhibiting mechanism of propamidine against botrytis cinerea now. At present, the inhibiting mechanism of propamidine against Botrytis cinerea Pers. will be studied using electron microscopy and biochemistry techniques. The main results and conclusions were given as follows:1. Effects of propamidine treatment on mycelial shape and ultrastructure of Botrytis cinerea PersoonThe studies revealed that propamidine not only inhibited the hyphal growth, but also caused a series of marked hyphae morphological and cell structural alterations. These changes are generally irregular swelling, excessive branching and many tumors on the surface of hyphale, considerable thickening of the hyphal cell walls, particularly at the hyphal tip region, increasing vacuoles and electrondense bodies, excessive mitochondrion irregular swelling, and so on. Above cytological change resulted in the necrosis of hyphal cell.Scanning electron micrographs displayed that the quantity of conidium decline and conidia collapsed practice of propamidine at the beginning of disease; don’t influence the quantity of conidium but conidia collapsed practice of propamidine after of disease. Transmission electron micrographs revealed that Botrytis cinerea Pers. Can’t invade tomato of epidermal organise practice of propamidine at the beginning of disease; but Botrytis cinerea Pers. Can invade tomato of epidermal organise collapsed practice of propamidine after of disease. propamidine can inhibited the hyphal growth, the tip of the hyphae were shrunken, considerable thickening of the hyphal cell walls, particularly at the hyphal tip region, cytoplasm inhomogeneity and inclusion in cell exosmosis, mitochondrion manifold, vesiculated hypha cell, cell empty and so on spray propamidine at various periods. Above cytological change resulted in the necrosis of hyphal cell. The studied showed that propamidine had distinct protective and therapeutic action influence.2. Effects of propamidine treatment on energy metabolize of Botrytis cinerea PersoonThe electric conductivity in Botrytis cinerea solution which was treated with different concentrations propamidine was measured using digital conductivity meter. We discovered that the electric conductivity gradually increased as the concentration of propamidine increased. The result compares with the observed outcomings usingelectron micrographs are very similar. These indicated that the structure of cell membrane was destroyed when Botrytis cinerea was treated with propamidine.Mitochondria, whose primary function is to produce energy, play a central role in cellular metabolism and in the control of bital functions. Mycelium respiration was first examined. It may be noted that the respiratory activity of treatment mycelia with different concentrations propamidine were less that of the control, but they have sameness of order of activity change. For the propamidine treatment, the State 3 respiration, state 4 respiration, respiratory control ratio and ADP/O ratio were significantly lower when compared to the corresponding controls at various contentration points.3. Effects of propamidine treatment on substance metabolize of Botrytis cinerea PersoonDecrease of respiratory activity or energy synthesize will changes the substance catabolism. To improve this, we measured the content of total sugar, reducing sugar, protein and fat of Botrytis cinerea treat with different concentrations propamidine. The results were shown as follows: 1) the content of total sugar, reducing sugar and protein initially increased and then decreased as the concentration of propamidine increased. 2)the content of fat always was increased when the propamidine concentrations was increased. 3) the content of total sugar, reducing sugar, protein and fat in Botrytis cinerea treat with different concentrations propamidine were more than the content of control’s. These results show that substance catabolism was inhibited when the Botrytis cinerea was treated with different concentrations propamidine.4. Effects of propamidine treatment on mitochondrial complexes activity of Botrytis cinerea PersoonTo examine how propamidine influences mitochondrial individual respiratory chain enzymes activities, mitochondria were isolated. The mitochondria of various concentration propamidine treatment were assayed indicidually to determine activity related to respiratory chain enzymes and oxidative phosphorylation. Activities from the excised control were used as the basis for comparison and set at 100%. Comparisons were first carried out to determine if there were significant changes in controls following treatment and, second, to determine if there were significant differentces between th control and the treatment at various contentration points. The results shows that the activity of mitochondrial respiratory chain enzymes have not been changed whether the Botrytis cinerea was treat with propamidine or not. This result indicated the propamidine did not combined with mitochondrial respiratory chain enzymes for changing their activity.When we first treated Botrytis cinerea with different concentrations of propamidine, and then extracted all of mitochondrial respiratory chain enzymes for detecting their activity. The results were shown as follows: 1)when Botrytis cinerea was treated with increasing concentrations of propamidine for one day, the activitys of complexⅠ,Ⅱ,Ⅳand ATP enzyme were increased. But the activity of complexⅢwas decreased . 2)when Botrytis cinerea was treated with increasing concentrations of propamidine for three days, the activitys of complexⅠ,Ⅱ,Ⅲ,Ⅳand ATP enzyme were decreased. These results indicated that the propamidine may be inhibited the activity of complexⅢ.In summarize, we can draw the conclusion about the inhibiting mechanism of propamidine against Botrytis cinerea. The propamidine will enter into the organism cell when the Botrytis cinerea was treated with propamidine. Then the propamidine will be combining with a gene region which encoded a subunit of complexⅢ, and inhibiting the expression of this gene. At last, the decreased activity of complexⅢwill be acrousing respiration weaken, energy synthesis lower, mitochondria increased and cell membrane broken. These changes will deform the mycelium shapes, control the growth.更多还原