【作者】 贾芝琪；

【导师】 李颖章；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 硕士

【摘要】 本实验以棉花抗病品种BD-18和感病品种泗棉三号的悬浮细胞为材料,研究了棉花细胞在黄萎病菌毒素处理后H₂O₂和NO的变化,并用半定量RT-PCR的方法检测毒素、H₂O₂和NO对棉花谷胱苷肽-S-转移酶（GST）基因转录水平的影响。 本实验首先确定了用冷冻浓缩后透析的方法初步提纯棉花黄萎病菌毒素,该方法能有效去除粗提毒素中的蔗糖和盐离子并能最大程度的保留黄萎病菌分泌的蛋白成分;对毒素蛋白成分鉴定结果表明蛋白条带多,每种蛋白的含量少,小分子量蛋白含量多,糖蛋白染色结果显示,毒素蛋白中分子量在35.8kDa-83.2kDa之间的蛋白大多数为糖蛋白,分子量小于35.8kDa的蛋白多为非糖蛋白;性质鉴定结果显示,毒素中有活性的蛋白成分具有部分可耐高温高压,且毒素蛋白中的蛋白成分和糖基成分都具有致萎活性。 用毒素处理抗病和感病的棉花悬浮细胞,可检测到H₂O₂和NO含量的瞬时升高,并且抗病品种显著高于感病品种。用SNP（NO供体）、H₂O₂、cPTIO、CAT处理棉花的悬浮细胞,结果发现,NO具有氧化与抗氧化双重作用,行使何种功能与浓度有关,在合适的浓度范围内具有抗氧化的作用。用cPTIO、CAT与毒素共同处理悬浮细胞结果说明在毒素处理过程中NO和H₂O₂两个信号分子互相影响。 半定量RT—PCR结果显示,毒素可增强GST基因的表达,抗病品种能对毒素处理做出快速反应;H₂O₂和NO均参与GST基因的表达,但可能是通过两条不同的途径。更多还原

【Abstract】 In this study, an attempt is made to account for the observed difference in the susceptibility of Gossypium hirsutum L. （cv. BD-18 and Simian 3, resistant and susceptible to V. dahliae, respectively） to toxin of Verticillium dahliae, by looking for differences in the timing and level of production of NO and H₂O₂, and their effects on the expression of GST gene.In this experiment, a new method was used to purify the toxin protein produced by Verticillium dahliae. The supernatant of fungus culture was frozen and dried with Lyophilizer first, and then dialysed by Dialysis Membranes （MWCO 1000） after dissolved in distilled water. This method can eliminate the salt and sucrose in culture medium and reserve the protein component almost completely. VD-toxin were analysed using sodium dodecyl sulfate polyacrylamide gel analysis（SDS-PAGE）. The results indicated that the protein components were very complex, and included glycoprotein within 35.8kDa-83.2kDa. Furthermore, treatments on glycoproteins with high temperature, conA and proteinase alleviated, not abolished, their activities.NO and H₂O₂ production were assayed in two cultivars of cotton cells which were treated with VD-toxin. The resistant cultivar exhibited higher induced levels of NO and H₂O₂ compared to the susceptible cultivar. The dual effects of NO were verified in this research. NO was either oxidative or antioxidative which depends on its concentration. NO took part in antioxidative system within appropriate concentration.GSTs are important enzymes in plants response to stress. We use SqRT- PCR to analyze the expression changes of GST gene in cotton suspension cultures. The result revealed that VD-toxin can induce the expression of GST gene, and the resistant cultivar exhibited higher and earlier induced levels compared to the susceptible cultivar. NO, H₂O₂ also induced the expression of GST gene, however NO induced defense responses against VD-toxin may independent on H₂O₂ signaling pathways leading to GST gene expression.更多还原