【作者】 李姝江；

【导师】 朱天辉；

【作者基本信息】 四川农业大学 ， 植物病理学， 2013， 博士

【摘要】 暗孢节菱孢菌是引起四川栽培区杂交竹梢枯病的新病原,其致病机理研究不深入,特别在该菌蛋白毒素方面的研究尚为空白。本论文以该病原为对象,在研究其蛋白毒素诱导因子基础上,对蛋白毒素进行分离纯化、组分和分子结构、基本性质的分析,探索蛋白毒素对杂交竹生理代谢影响规律,弄清其作用浓度临界值,比较病菌与蛋白毒素对杂交竹伤害的生理学差异；并通过毒素免疫化学方法标记分析蛋白毒素结合位点和探索蛋白毒素对杂交竹嫩枝离体线粒体生物物理特性及呼吸作用的影响。获得以下主要研究结果：1.在采用单因素筛选最佳培养基及其成分的基础上,运用正交试验确定诱导暗孢节菱孢产蛋白毒素的温度、时间、pH、光照、瓶装量、接种量六个因素,从而得出最优诱导方案为：在改良Fries培养基+杂交竹煎汁为基础培养基中,乳糖等量代替葡萄糖；温度25℃,pH7,黑暗条件下,1块菌丝块（5mm）接种于80mL培养液（300mL三角瓶）振荡培养15d。在此诱导方案下,梢枯病菌产毒能力显著提高,杂交竹嫩枝感病指数在96h时高达85.69%。2.采用硫酸铵分级沉淀法对暗孢节菱孢的发酵上清液进行盐析,生测结果显示上清液没有活性,沉淀有活性,其盐析的最适饱和度为50%。此粗提物经SephadexG-50分子筛层析、High Q Sepharose Fast Flow强阴离子交换层析和Sephadex G-75分子筛层析后获得1个活性峰,SDS-PAGE电泳检测该峰为单一条带,以相对迁移率（mR）算得该蛋白的分子量为34.5kDa。将该峰进一步用RP-HPLC的方法进行纯度检验,经洗脱后得到2个峰,活性检测表明出峰时间为21min的峰2具有致萎活性,并命名为AP-toxin。采用Edman降解法成功测定了AP-toxin的N端13个氨基酸序列,即H2N-Pro-Pro-Ser-Gln-Val-Gln-Arg-Ala-Pro-Glu-Leu-Thr-Ser。经NCBI蛋白质数据库比对分析,推断该蛋白毒素与Phytophthora sojae的hypothetical protein PHYSODRAFT₅63177氨基酸残基序列100%同源。对AP-toxin’性质研究的结果表明,该毒素耐温度范围为0-80℃、耐酸碱的范围为pH4-10、耐白炽光却不耐紫外线照射,对蛋白酶K和胰蛋白酶具有较好的耐受性,有一定专化性、介于专化性与非专化性毒素之间。3.采用针刺法对AP-toxin进行田间致病力检测,结果显示不同浓度AP-toxin处理的杂交竹针刺处均出现不同大小的褐色菱形病斑,与用A.phaeospermum菌悬液处理的一致,但反应快于病原菌；40、80μg/mL AP-toxin处理从15d开始与A.phaeospermum菌悬液处理的差异不显著。在不同杂交竹品种中,AP-toxin有效起始作用浓度不同：对抗病品种（3号和6号杂交竹）的有效起始作用浓度为10-20μg/mL；对感病品种（8号和30号杂交竹）的有效起始作用浓度为5-10μg/mL。4.采用浸渍法测定AP-toxin对4个杂交竹品种生理代谢的影响。毒素处理后,酚代谢的总酚和类黄酮含量先上升后下降,大小顺序均为6#>3#>30#>8#；核酸代谢中的总核酸、DNA、RNA含量均下降且DNase和RNase活性增加,但抗病品种中二者的降幅和增幅小于感病品种；蛋白质代谢中的可溶性蛋白含量先上升后下降,抗病品种增幅明显大于感病品种,4个品种蛋白酶活性均升高；糖代谢中4个品种可溶性糖含量均呈显著下降趋势,但6#>3#>30#>8#,但还原糖含量变化规律相反,均不同程度升高,且大小顺序为8#>30#>3#>6#。同时,测定了毒素对杂交竹防御酶系（POX、 SOD、PAL、PPO、几丁质酶、p-1,3葡聚糖酶）活性的影响,抗病品种的POX活性先升高到峰值然后下降后又上升,后期趋于稳定；感病品种均显著下降。SOD、PAL活性动态变化的规律均为初期升高而后期下降的趋势,大小顺序为6#>3#>30#>8#。抗病品种PPO活性0-72h大幅增加,之后下降保持一定水平；感病品种在36h时达到峰值,随即下降,在96h已低于无菌水对照组。4个品种几丁质酶活性均在0-36h内显著升高,但感病品种和抗病品种间差异显著。抗病品种p-1,3葡聚糖酶活性在0-240h内该酶活性迅速升高,至240h均达到峰值,之后有所下降但始终高于初始阶段；感病品种在整个测试期内其酶活均缓慢下降,但8号下降趋势更为明显。5.结合AP-toxin处理后杂交竹的症状表现及感病指数,以及AP-toxin伤害与杂交竹生理代谢的相关性分析,结果显示,总酚和类黄酮含量与抗性显著相关,而与感病指数显著负相关,但类黄酮含量与时间不相关；核酸代谢的五项指标与时间和抗性均显著相关,总核酸含量和RNA含量与感病指数呈显著负相关；蛋白质代谢中的蛋白酶活性与感病指数相关性不显著外,其余均达到显著水平；可溶性糖含量与时间、抗性、感病指数相关性达到极显著水平,而还原糖除与抗性极显著负相关外,与时间和感病指数相关性不显著。在6种防御酶活性中,与抗性呈显著或极显著正相关,而与感病指数呈负相关,但仅POX和β-1,3葡聚糖酶与时间显著正相关。另外,感病指数与时间呈极显著正相关,与品种抗性之间为极显著负相关。6.以AP-toxin免疫新西兰白兔制备毒素特异性抗血清,并将该蛋白用不同浓度的抗体吸附后处理杂交竹幼嫩枝条。结果表明,毒素所引起的症状有不同程度的减轻,说明所制备的抗体在与毒素发生特异性免疫学反应的同时,可部分封闭毒素分子上与毒素受体结合的位点;利用竞争酶联免疫吸附试验（ELISA）测定杂交竹嫩枝的质膜制剂与毒素蛋白的结合活性显示,质膜制剂与毒素结合后能部分阻断毒素与其抗体的免疫学反应,即质膜制剂中含有毒素的结合位点,且不同品种的结合活性有差异；胰蛋白酶和加热处理质膜制剂后,质膜制剂对毒素与其抗体反应的抑制作用消失,证实质膜制剂中与毒素结合的是蛋白类物质。7.采用不同检测技术（如荧光偏振法、中性红法、氧电极法等）测定AP-toxin对杂交竹嫩枝线粒体膜的流动性、表面电位、肿胀度、聚集度及脂质过氧化物MDA、 H2O2、辅酶Q10（COQ1O）含量、线粒体呼吸功能的影响。结果表明,蛋白毒素胁迫使线粒体膜流动性减弱、表面电位减低、肿胀度增大、聚集度降低；CoQ10含量下降,H2O2、MDA含量的增加,细胞色素C氧化酶（CCO）、ATPase活性亦下降,呼吸控制率（RCR）和磷氧比（P/O）明显降低,说明蛋白毒素对杂交竹嫩枝线粒体膜造成损伤,线粒体脂质过氧化程度增加,膜完整性被破坏,呼吸作用受抑制。更多还原

【Abstract】 Arthrinium phaeospermum is the new pathogen causing Bambusa pervaiabilis xDendrocalamopsis daii blight in Sichuan cultivating area, whose pathogenic mechanism is still in infancy, especially in protein toxin produced from this pathogen is also lack. Based on the induced factors of protein toxin produced from this pathogen, the protein toxin was isolated and purified, its component, molecular structure and basic characteristic were analysed. The influence of protein toxin on the physiological metabolism of B. pervaiabilis×D. daii was explored, the critical value of activated concentration was clarified, and the physiological difference in damage of B. pervaiabilis×D. daii between the protein toxin and the pathogen. The binding site of the protein toxin was analysed by the immunochemical method to label toxin, and the effects of the protein toxin on biophysical characteristic and respiration of the mitochondrion of B. pervaiabilis×D. daii shoot were explored. The results were as follows:1. On the basis of screening the optimum culture medium and culture composition by the univariate analysis, the temperature, time, pH, light, bottle volume and inoculated amount of inducing the protein toxin produced from A. phaeospermum were comfirmed by orthogonal test. The optimal inducing scheme was including as below:in modified Fries medium+B. pervaiabilis×D. daii shoot juice, the glucose was replaced with the equal amounts of lactose; one mycelium mass （5mm） was inoculated into80mL culture medium in300mL triangular flask （pH7） and shaking cultured15d at25℃in dark condition. Under this sheme, the ability of produced toxin of A. phaeospermum enhanced siginificantly, and the disease index of B. pervaiabilis×D. daii shoot was85.69%at96h.2. The fermented supernatant of A. phaeospermum was salted out by ammonium sulfate precipitation, the bioassay results showed that the supernatant liquid had not activity, the precipitation had activity, and the optimum saturation was50%. One active peak was obtained by Sephadex G-50chromatography, High Q Sepharose Fast Flow chromatography and Sephadex G-75chromatography, which was one single band tested by SDS-PAGE analysis, and the molecular weight was34.5kDa calculated by the relative move rate （mR）. The two elution peaks were further collected for purity of RP-HPLC, the activity analysis indicated that peak2with the peak time of21min had wilting acitivity, and it was named as AP-toxin. The sequencing result of AP-toxin showed13amino acids sequence of N-terminal end from the beginning was H2N-Pro-Pro-Ser-Gln-Val-Gln-Arg-Ala-Pro-Glu-Leu-Thr-Ser, which had100%homology with the hypothetical protein PHYSODRAFT₅63177from Phytophthora sojae by the homophyly retrieval in NCBI. Characteristics of AP-toxin showed that this toxin temperature-resistant range was0-80℃, acid-base resistant was pH4-10, as well as it could resist incandescent light but not resist ultraviolet ray. In addition, it had stability against protease K and trypsin, and had some specialization which meant it was between the specialization and non-specialization toxin.3. The puncture method was used to assay the pathogenicity of AP-toxin in field, the results showed that the different sizes of brown diamond disease spots appeared in the acupuncture places of bamboos treated with different concentration of AP-toxin, as the same as the treatment of Aphaeospermum suspension, although the reaction was quicker than the pathogen. AP-toxin treatments of40and80μg/mL had no significant difference with the pathogen treatment from15d. In different varieties, the dose-response concentrations were different:the dose-response concentration of5-10μg/ml for the resistant varieties （No.3and No.6）, and10-20μg/ml for the susceptible varieties （No.8and No.30） were observed.4. The effects of AP-toxin on the physiological metabolisms in four bamboo varieties were determinated by the impregnation method. After treated by toxin, the total phenol and flavonoid contents in phenolic metabolism increased and then decreased, the order was6#>3#>30#>8#. Total nucleic acid, DNA, and RNA contents in nucleic acid metabolism decreased but DNase and RNase activities increased, the decline and increase amplitudes of resistant varieties were less than the susceptible varieties. The soluble protein contents increased first and then decreased, the increase amplitude of resistant varieties was significantly more than the susceptible varieties, and the protease activity in four varieties rose. The soluble sugar contents in four varieties significantly declined, the order was6#>3#>30#>8#; but the change rule of reducing sugar was opposite, which had different degree of increase, and the order was8#>30#>3#>6#. Moreover, the effects of toxin on the activities of the defense enzymes （POX, SOD, PAL, PPO, chitinase and β-1,3-glucanse） were determined. POX activity of resistant varieties increased first until the peak value and then decreased and increased, and maintained the stability at the later period; but the susceptilble varieties significantly decreased. The dynamic change trends of SOD and PAL activities were initial increase and the later decrease, the order was6#>3#>30#>8#. PPO activities of resistant varieties increased drastically during0to72h followed by a decrease and then remained stable; the activities of suscepitible varieties reached their peaks at36h followed by a decrease, and the values at96h were lower than the respective control. Chitinase activities in four bamboo varieties increased dramatically and reached a relatively high value at36h, but the difference between suscepitible varieties and resistant varieties was significant. β-1,3glucanse activity in the resistant varieties showed a growing trend until activity peak formation at240h, which was followed by a decline; the activities in the susceptible ones declined in the whole testing period, particularly in No.8.5. Combined with the symptom and disease index of B. pervaiabilis×D. daii treated by AP-toxin, the correlation analysis between damage of AP-toxin and physiological metabolisms of bamboo, which indicated that the correlation of total phenol and flavonoid contents was significant to the resistance, but negatively significant to disease index, the correlation between flavonoid content and time was not significant. The nucleic acid metabolism had significant correlation with time and resistance, total nucleic acid content had negative correlation with disease index. Except the correlation between protease activity and disease index was not significant, the others were significant in protein metabolism. The correlations between soluble sugar content and time, resistance, disease index were remarkably significant, but it was remarkably negatively significant between reducing sugar content and resistance, and was not significant to time and disease index. The correlations of defense enzymes were significant or remarkably significant to the resistance, and were negative significant to disease index, but only POX and P-1,3glucanse activities had positively significant correlation with time. In addition, the disease index had remarkably positively significant correlation with time, and remarkably negatively significant correlation with the reisistance. 6. The specific antiserum against the toxin was prepared by injecting the New Zealand white rabbit with AP-toxin. The results showed that the symptom was alleviated to different degree when the toxin absorbed by different concentration antibodies was inoculated B. pervaiabilis×D. daii shoot, illuminating that the prepared antibody not only immunologically reacted with the toxin, but also partly blocked some sites of toxin molecular which could recognize the binding sites on the hybrid bamboo cells. The binding activity of plasmalemma preparation of tender branches with protein toxin was determined by a competitive enzyme-linked immunosorbent assay （ELISA）. The results revealed that the immunological reaction of the toxin and the antibody could be inhibited by the plasmalemma preparation, which hinted that the plasmalemma preparation contained the binding sites of the toxin. But the binding activities of different varieties had different. After treated by trypsin or heating, the plasmalemma preparation could not inhibit the immunological reaction of the toxin and the antibody, which verified the protein in the plasmalemma preparation was responsible for binding with the toxin.7. The effects of AP-toxin on the membrane fluidity, surface electronic potential, swelling, aggregation and the contents of lipid peroxide substance MDA, H2O2, Coenzyme Q10（CoQ10）, and respiration function of the mitochondrion in B. pervariabilis×D. grandis shoot were determined by the different detection techniques （fluorescence polarization method, neutral red method, oxygen electrode method, etc.）. The results showed that the protein toxin made the membrane fluidity weakening, surface electronic potential and aggregation decrease, swelling increase. It also made CoQ10content decrease, the contents of H2O2and MDA increase, the activities of cytochrome c oxidase （CCO） and ATP decrease, respiration control rate （RCR） and oxidative phosphorilation ratio （P/O） having significant reduction. The above results demonstrated that the protein toxin might damage the mitochondrial membrane, the lipid peroxidation of mitochondrion increased, the membrane integrality was destroyed, and the respiration was inhibited.更多还原