【作者】 李建刚；

【导师】 郭坚华；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 细菌性青枯病是一种由青枯劳尔氏菌(Ralstonia solaanacearum, E F Smith)引起的毁灭性土传病害,发病植物茎叶萎蔫下垂直至全部枯死,是世界上危害最大、分布最广、造成损失最严重的植物病害之一。青枯劳尔氏菌R. solaanacearum可侵染200多种植物,其中包括生产中重要的作物,如番茄、马铃薯、烟草、花生和香蕉。青枯病菌是一个复杂的群体,有明显的生理分化,不同地区和不同寄主来源的菌株,在寄主范围、致病力、生化型、血清型等细菌学特性上差异很大,因此增加了对此病害防治的难度。近几年来许多科研工作者从不同角度、用不同方法对青枯劳尔氏菌进行了多方面的研究。Harpin蛋白是由革兰氏阴性植物病原细菌Hrp (hrp, hypersensitive response and pathogenity)基因簇中hrp基因编码的、性质和功能相似的一类蛋白质,富含甘氨酸,缺少半胱氨酸,对蛋白酶敏感,对热稳定,大多对寄主植物具有毒性,导致寄主发病,能在非寄主植物上引起过敏反应。本研究以青枯劳尔氏菌(Ralstonia solanacearum) ZJ3721的基因组DNA为模板,成功克隆了全长为1143bp的popW基因以及popW(1-159)和popW(160-366)两个基因片段,并把这三个基因连接到表达载体pET30a(+)进行了原核表达。在1mM异丙基硫代-β-D-半乳糖昔的诱导下(IPTG),这三个基因都能在Escherichia coli BL21(DE3)中成功表达。通过对PopW表达条件的优化发现IPTG的最佳浓度为0.01 mM。运用镍柱纯化获得了高纯度的PopW融合蛋白。另外运用同源重组的方法我们还构建了青枯劳尔氏菌popW基因的插入突变体ZJ3722。我们从青枯劳尔氏菌ZJ3721中鉴定了一个39.79 KDa的新胞外蛋白PopW,它同以前报道的harpin类物质具有相同的性质,酸性,富含甘氨酸和丝氨酸,缺少半胱氨酸并且是一种耐热蛋白。能在非寄主植物烟草上引起过敏性反应。在一级结构上,PopW是由两个结构域组成,包括N-末端159个氨基酸的harpin结构和C-末端207个氨基酸组成的果胶酶的结构域,他们之间由富含甘氨酸和丝氨酸的短肽连接起来。PopW的harpin结构域也能引起烟草发生HR反应。虽然PopW具有果胶酶结构域,但并没有果胶酶活性。另外,我们从其它十九个不同遗传多样性的青枯劳尔氏菌中都扩增到了popW基因,这说明popW是广泛存在于青枯劳尔氏菌的。把popW和绿色荧光蛋白(GFP)构成融合基因,连接至植物转基因载体pBI121,在土壤杆菌的介导下转入洋葱表皮细胞。荧光显微镜观察结果表明,PopW蛋白是定位在植物的细胞壁上。因此,我们推测PopW在青枯劳尔氏菌与寄主互作的过程中起帮助定殖的作用。通过popW的缺失突变体菌株的接种实验发现popW对非寄主植物烟草的HR反应和寄主植物的致病过程中不起决定作用。综上所述,PopW (39.79 KDa)是青枯劳尔氏菌中广泛存在的一个定位于寄主细胞壁上,受hrpB基因的调控,通过三型泌出系统分泌到胞外的且不影响菌株对寄主致病性的新harpin。对青枯劳尔氏菌(Ralstonia solanacearum) ZJ 3721中表达的PopW蛋白诱发烟草系统获得抗性进行了研究。以25 gg／mL PopW溶液处理三生烟草叶片24 h后,在处理叶片及其上、下各2片叶片接种TMV,结果显示处理叶及其上、下各2片叶片上的病斑数显著少于只接种水和TMV的对照叶片。同时PopW处理的烟草叶片中还伴随着H202的迸发,在处理后24h时,累积量达到了最大为1.972μM／gFW。另外在此过程中,烟草中还有防御酶系PAL,POD和PPO的时序变化,PAL酶活在12h达到了峰值1215.33U／g,POD和PPO在24h和10h到达了最大值,分别是1110.5U／g和863.67U／g。PopW还能诱导野生型烟草和拟南芥中PR-1基因的表达,而二者转nahG基因的突变体植株却没有该基因的表达。所以PopW能够诱导植物产生系统抗性,并且该抗性属于水杨酸介导的SAR。茄子黄萎病是一种严重的土传维管束病害,本研究发现PopW蛋白能够诱导茄子产生抗性从而防止黄萎病菌的侵染。同时我们从353株茄子根围细菌中筛选到一株产几丁质酶的生防细菌CH2。根据16S rRNA基因序列和生理生化实验确定其为蜡状芽孢杆菌。它能在以几丁质为唯一碳源的培养基上生长,经SDS-PAGE和特异性底物验证确定其可以向胞外为分泌15KDa的几丁质酶。通过平板实验我们发现,该菌的悬浮液、上清液及其产生的浓度为0.005%几丁质酶液可以有效的抑制黄萎病菌分生孢子的萌发。该菌产生几丁质酶的最适pH值是7.1,最佳温度为40℃。在温室试验中,CH2菌悬液的防效可达69.69%,25μg／mL的PopW蛋白溶液,CH2上清液及0.01%的几丁质酶液的防效分别为57.46%,54.04%和53.13%。所以CH2菌株具有很好的防治茄子黄萎病潜力。更多还原

【Abstract】 Ralstonia solanacearum is a major soil-borne plant pathogen in subtropical and tropical areas that naturally infects roots and multiplies in xylem vessels which has an unusually wide host range since plant species susceptible to the pathogen have been observed to occur in more than 200 plant species belonging to approximately 50 botanical families. Huge economic losses are caused annually by this pathogen in China and many other countries R. solanacearum is a very complex community with dishtingushed physiological characterization. It is difficult to control because strains from different regions and host have significant difference in pathogenicty, biovar and serology. Many scientists have done some researchs about R. solanacearum using many methods from different aspects.Harpins are extracellular proteins encoded by Gram-negative bacterial hrp (hypersensitive response and pathogenity) gene cluster. They are a major class of proteins that travel the pathway, elicit the HR (hypersensitive reaction) when infiltrated into the apoplast of leaf tissue. They are heat stable, rich in Gly and/or Ser, lack Cys, and differ in their primary sequences.popW(1143 bp in length) and its dervivates,popW(1-159)and popW(160-366) were cloned from Ralstonia solanacearum ZJ3721. The three genes were liagased to expression vector pET30a (+) and expressed successfully in Escherichia coli BL21 (DE3) induced by 1mM IPTG. The optimum concentration of IPTG for the expression of PopW is 0.01 mM. The fused PopW protein was purified from supernatant of its host by Ni column. Finally, the popW-deficient mutant of Ralstonia solanacearum was construced by homolougous recombination.This study identified a new extracellular bacterial protein, PopW, from R. solanacearum ZJ3721. PopW, like other known harpins, is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into plants, PopW induced rapid tissue collapse on tobacco (non-host). This hypersensitive reaction (HR)-eliciting activity of PopW was heat stable and protease sensitive. PopW of R. solanacearum consists of two domains connected by a Pro-and Ser-rich sequence. The N-terminal domain (amino acids 1 to 159) alone was able to elicit the HR, while its C-terminal region (amino acids 160 to 366) is homologous to pectate lyases (PLs). Neither this C-terminal fragment nor the full-length PopW showed PL activity. PopW was widely distributed in other R. solanacearum strains from different genetic diversity by PCR amplification identification. The subcellular location analysis via transfering popW and GFP fused gene to onion epimdermal meideated by Agrobacterium tumefaciens revealed that PopW targeted plant cell wall. PopW did not affect the virulence of R. solanacearum because popW-deficient mutants retained the wild-type ability to elicit HR on non-host plants and to cause disease on tomato host plants. Thus, we concluded that PopW is a cell-wall associated,hrpB-dependent,39.79 kilodaltons new harpin which is widely distributed in different R. solanacearum strains and has no influence on the pathogenicity of this pathogen.We have investigated the tobacco systemic acquire resistance induced by PopW protein from R. solanacearum. Nicotiana tobacum cv. Sam sun NN tobacco leaves were treated by 25μg/mL PopW followed by the infection of TMV onto these leaves, and the upper and lower two leaves of each one. The Lesion number infected by TMV is significantly less than blank control treated with sterile water and TMV. H2O2 bursted in the leaves sprayed by PopW, which reached the maximum of 1.972μM/g FW (fresh weight) 24h after the tobacco was treated. The activities of defence enzyme (PAL, POD and PPO) were also changed. The activity of PAL reached the climax of 1215.33 U/g 12h after treatment. POD and PPO achieved the peak activities 10h and 24h after treatment, which were 863.67 U/g and 1110.5 U/g, separately. The PR-1 gene in tobacco and Arabidopsis were up-expressed after PopW treatment, while the PR-1 did not express in transgenic nahG tobacco and Arabidopsis. In conclusion, plant resistance induced by PopW is systemic acquired resistance meditaed by salicylic acid.Verticillium wilt on eggplant is a devastating soilbrone disease. PopW was found effectively inhibiting Verticillium daliae infection by inducing eggplant acquired resistance. A chitinase-secreting strain CH2 was selectef from 353 strains isolated from rhizosphere of eggplant. This strain was identified to be of Bacillus cereus based on 16S rRNA gene sequence alignment and biochemical and physiological characteristics. The strain can live on chitin-Ayers (CA) medium which only has chitin as the carbon resource, the evaluation of its activity, combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), showed it can secreted a 15.0 KDa chitinase. On glass slides, germination of the fungal spores was effectively suppressed by the bacterial suspension, supernatant from the suspension, and 0.005%solution of chitinase extracted from the strain CH2. The optimum pH for chitinase was 7.1 and optimum temperature was 40℃. At that temperature, high-level chitinolytic activity was retained for 10 days. In greenhouse experiments, suspension of the cells of the CH2 strain reduced the severity of Verticillium wilt on eggplant by 69.69%,25μg/mL PopW solution, CH2 supernatant by 57.46 and 54.04%, and the enzyme diluted to 0.01% strength by 53.13% in 14 days. Strain CH2 and its chitinase have good commercial potential in controlling Verticillium wilt.更多还原