【作者】 王辉；

【导师】 严海燕；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 青枯菌为青枯病病原菌,为革兰氏阴性菌。其侵染植物达44个科,300多种植物,以茄科最多。其分类系统很多,国际公认的分类系统有两种:一是根据寄主范围划分,分为5型;二是根据菌株对3种二糖（麦芽糖、乳糖和纤维二糖）和3种己醇（甘露醇、山梨醇和卫矛醇）氧化产酸能力划分,划分为5个变种。其分泌系统可以划分为6种类型。青枯菌的主要毒性因子为胞外多糖和胞外蛋白。青枯病之所以难于防治,在于青枯菌基因组的可塑性。青枯菌的基因组具有双组分结构（a bipartite genome structure）、替换密码子使用区域（alternate coden usage region, ACUR）和插入子和转座子（insertion sequences）。此外,青枯菌的基因组还有基因水平转移（horizontal gene transfer, HGT）的发生。花生青枯病是茄劳尔氏菌引起的一种细菌性维管束病害,中国、印度尼西亚、越南等是这一病害危害较重的国家,其中我国的受害面积最大。发病高峰期发病率达30%以上,甚至绝收。青枯病的防治方法很多,如利用抗病品种、利用转基因植物、合理轮作、化学防治和生物防治等。选育抗病品种被公认为是防治花生青枯病最经济有效的方法。青枯菌侵染植物的过程,涉及到的防御系统复杂,从不同的角度可分为预存抗性与系统抗性,非寄主抗性与寄主抗性,及苗期抗性与成株抗性等。培育筛选抗病品种和利用转基因技术培育抗青枯病品种仍是当前防治青枯病经济有效,简便易行的途径。植物的抗病过程,广泛涉及到了植物次生代谢,主要可以分为2类,一类是抗毒素,是组成型表达的物质。一类是植保素,是微生物侵染后,被诱导才表达的物质。植物的抗性机制是植物在与病原菌的长期斗争中存活的根本,并随着病原菌不断进化而协同进化。植物的氧化还原的酶类,正是植物次生代谢反应的参与者,与植物的抗性密切相关。PAL参与了植物色素形成,细胞分化和木质化作用,植物抗病作用和次生代谢产物调控等。其中,次生代谢产物调控包括调控黄酮类化合物合成,调控紫杉醇合成,调控生物碱合成和调控红厚壳素等其他次生代谢产物合成。CAT的主要功能是清除H₂O₂,并转化为无毒的H₂0和0₂。自由基的危害很大,可导致生物活性分子被氧化,故CAT对减轻机体损伤的意义很大。PPO参与了植物的光合作用、抗病虫害、生长发育,花色的形成,防御系统及伤口愈合等。POD参与木质素和木栓质的合成,参与活性氧代谢过程,参与生长素的降解,参与植物对外界不良胁迫的应答和植物组织褐变等。本研究将青枯菌菌液制备成OD600为0.5和0.1两浓度。土培‘远杂9102’（抗病）和‘中花8号’（感病）两品种花生至三叶期,然后于主茎第一分蘖处下方注射20μl已制取的青枯菌菌液。分别于接种后的第5、7、10、12和14天取样,取样部位为主茎第一分蘖处到第二分蘖处之间的茎。并对各样品测定了PAL、CAT、PPO、POD的酶活性和可溶性蛋白质含量。通过对抗病品种和感病品种的4种酶活性的对比,发现前期抗病品种酶活性不是都高于感病品种,后期,抗病品种的酶活性则都高于感病品种。结果显示,花生抗病品种和感病品种在感染青枯病后期关键酶的酶活性与花生的抗病能力呈正相关关系。这一结论可作为鉴定花生抗病能力的参照指标之一。更多还原

【Abstract】 Ralstonia solanacearum is the bacterial wilt pathogen and Gram-negative bacteria. Plants which can be infected by Ralstonia solanacearum cover 44 families, more than 300 species. Solanaceae are the most. Mutiple classification systems of Ralstonia solanacearum exist, two of them are internationally accepted classification systems.: First, Ralstonia solanacearum is divided into 5 types according to host range; Second, 5 variants are divided into based on oxidation capacity using 3 disaccharides（maltose, lactose and cellobiose） and 3 hexanols（mannitol, sorbitol and alcohol Euonymus）. Its secretion systems can be divided into 6 types. The major virulence factors of Ralstonia solanacearum are extracellular polysaccharide and extracellular proteins. Because of plasticity of the genome of Ralstonia solanacearum, bacterial wilt is difficult to control. Genome of Ralstonia solanacearum has bipartite genome structure, alternate coden usage region, insertion sequences and transposon. In addition, horizontal gene transfer can happen in he genome of Ralstonia solanacearum.Peanut wilt is a bacterial vascular bundle disease caused by Ralstonia solanacearum. Severe disease hazards happened in China, Indonesia, Vietnam, etc. Affected areas of China are the largest. The incidence rate reaches 30% in the peak period, even 100%.There are many control methods on bacterial wilt, such as the use of resistant varieties and transgenic plants, proper rotation of crops, chemical control and biological control, etc. Breeding resistant varieties widely considered is the most cost-effective method on peanut wilt.Defense systems involved in the process of Ralstonia solanacearum infecting plants are complex. From different angles, these defense systems can be divided into preformed resistance and systemic resistance, non-host resistance and host resistance, seedling resistance and adult plant resistance, etc.The process of plant disease resistance widely Involves plant secondary metabolism, which can be mainly divided into 2 categories, one is antitoxin, which is expressed constitutively, The other one is phytoalexin, which is expressed after microbial infection.The resistance mechanisms of plants is the root cause that plants can survive in the long struggle with plant pathogens, and can coevolve with the pathogen constantly. Cultivating and screening of resistant cultivars and cultivating Ralstonia solanacearum resistant cultivars by transgenic technology are still the cost-effective, user-friendly way to control bacterial wilt.Redox enzymes of plants are participants of plant secondary metabolic reactions, and closely related with plant resistance. PAL is involved in plant chromogenesis, cell differentiation, lignification, disease resistance and regulation of secondary metabolites, etc. Regulation of secondary metabolites includes regulations of compounds of flavonoids compounds, paclitaxel, alkaloid and kalofilum kathing, etc. CAT’s main function is to disintegrate H₂O₂, and transform it into H₂0 and 0₂ which are non-toxic. Free radical is seriously harmed, and result in bioactive molecule oxidized, so CAT is very meaningful to reduce body injury. PPO is involved in plant photosynthesis, resistance to pests and diseases, growth, color formation, defense system and wound healing, etc. POD is involved in lignin and suberin synthesis, reactive oxygen species metabolic processes, the degradation of auxin, plant responses to external stress and plant tissue brown stain, etc.two concentrations OD600 of 0.5 and 0.1 was prepared by Ralstonia solanacearum suspension in this study.’9102’（resistant cultivar） and‘Zhonghua 8’（susceptible cultivar） were cultivated to the stage of three leaves by Soil, then were injected 20μl Ralstonia solanacearum suspension under the first tiller of the main stem. They were sampled on the fifth, seventh, tenth, twelfth and fourteenth day between the first and second tiller of the main stem. PAL, CAT, PPO and POD activities and soluble protein content of every sample were measured.4 enzymatic activities were compared. Not all of the 4 enzymatic activities of resistant cultivars were higher than that of susceptible cultivars in earlier stage, but all of enzymatic activities of resistant cultivars were higher than that of susceptible cultivars in later stage.The results showed that key enzymes activity of resistant and susceptible cultivars of peanut at later stage of bacterial wilt were positive correlated to peanut disease resistance. This conclusion can be a indicator as reference of identifying peanut disease resistance.更多还原