【作者】 毕朝位；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 微管是构成真核生物细胞骨架的主要成分,在细胞的生命活动过程中起重要作用。α-和β-微管蛋白是构成微管的主要成分,而β-微管蛋白也是多菌灵等苯并咪唑杀菌剂的作用靶标,其特定位点的突变将导致苯并咪唑杀菌剂抗性。禾谷镰孢菌是我国引起小麦赤霉病的主要病原菌,多菌灵是防治该病害的主要药剂之一,但多菌灵对禾谷镰孢菌的作用表型及禾谷镰孢菌对多菌灵的抗性表型与其它植物病原真菌有差异。本论文通过观察多菌灵对禾谷镰孢菌的细胞学尤其是有丝分裂的影响,明确多菌灵对禾谷镰孢菌的作用机制；通过研究禾谷镰孢菌两个β-微管蛋白基因的敲除突变体、与GFP的融合表达突变体的形态学和生理学表型、两者在菌体不同阶段的表达差异以及两个β-微管蛋白在细胞内的定位,从而明确禾谷镰孢菌两个β-微管蛋白基因的关系以及在禾谷镰孢菌生命活动过程中的作用,确定多菌灵对禾谷镰孢菌的作用靶标及禾谷镰孢菌对多菌灵抗性的机理,从而为禾谷镰孢菌的抗药性治理以及以新的微管蛋白靶标药剂的设计提供重要的理论依据。1禾谷镰孢菌分生孢子萌发过程中核相变化及有丝分裂过程观察本研究通过Giemsa染色观察禾谷镰孢菌分生孢子萌发过程中的核相变化及有丝分裂过程。观察表明,分生孢子细胞为单核,细胞核在分生孢子细胞内分裂后进入芽管,在芽管内进行多次分裂,使芽管内细胞核数目不断变化。禾谷镰孢菌有丝分裂过程可以分为4个时期,前期染色体逐渐浓缩变短,中期染色体清晰可见,后期染色单体发生分离并向相反的两极移动,末期形成新的子核。有丝分裂过程中染色体的分离同步或不同步,不同步分离中的滞后染色体形成后期桥的现象更为普遍。2多菌灵对禾谷镰孢菌分生孢子萌发和有丝分裂的影响已有研究结果表明多菌灵对灰葡萄孢菌Botrytis cinerea等多种真菌的作用机制是抑制有丝分裂,其β-微管蛋白发生突变是抗药性产生的原因。本研究通过比较多菌灵对禾谷镰孢菌和灰葡萄孢菌分生孢子萌发以及幼殖体内有丝分裂的影响,以探明多菌灵对禾谷镰孢菌的作用机制以及禾谷镰孢菌对多菌灵的抗性机制。禾谷镰孢菌野生型(多菌灵敏感)菌株的分生孢子在多菌灵处理条件下萌发产生的幼殖体发生扭曲、分支增多、伸长受到抑制；其分生孢子在无药剂条件下产生的幼殖体在多菌灵处理条件下产生相同的特征。在药剂处理条件下细胞核不能正常地进行有丝分裂,染色体呈散乱分布。同时,染色体有丝分裂指数(CMI值)在药剂处理后60min内很快升高,然后又快速下降。多菌灵处理灰葡萄孢菌野生型菌株产生相同的形态学和细胞学特征。灰葡萄孢菌多菌灵抗性菌株在药剂处理条件下能进行正常的萌发、生长及有丝分裂。但是,禾谷镰孢菌多菌灵抗性菌株在药剂处理条件下分支增多、CMI值有显著的变化,尽管未见有丝分裂受到明显的影响。以上研究结果表明,同灰葡萄孢菌一样,多菌灵对禾谷镰孢菌的作用机制也是抑制其有丝分裂,但两者的抗性机制有差异。3多菌灵对禾谷镰孢菌的作用靶标本研究分别敲除禾谷镰孢菌的β1、β2-微管蛋白基因,并测定各敲除突变体菌株对多菌灵的敏感性,结果表明,敲除其中任意一个β-微管蛋白基因的突变体菌株都对多菌灵敏感,但敏感性有差异。同时获得绿色荧光蛋白GFP基因与禾谷镰孢菌的β1、β2-微管蛋白基因的融合突变体菌株,通过荧光显微观察多菌灵对禾谷镰孢菌β1、β2-微管蛋白的敏感性差异,结果同样表明两β-微管蛋白与多菌灵的敏感性有差异。以上研究结果表明禾谷镰孢菌的β1、β2-微管蛋白都是多菌灵等苯并咪唑杀菌剂的作用靶标,但两β-微管蛋白与多菌灵的敏感性有差异,这种差异可能是由于两者240位氨基酸的不同而导致的。同时,研究还表明禾谷镰孢菌荧光蛋白标记的β-微管蛋白基因可以同源替换内源的同一β-微管蛋白基因,甚至两个内源β-微管蛋白基因都可以同时替换,并能正常表达,且对目标蛋白的生物学功能没有影响。4禾谷镰孢菌两个β-微管蛋白基因功能本研究通过测定禾谷镰孢菌β1、β2-微管蛋白基因敲除突变体菌株的营养生长特性、生殖生长能力、细胞有丝分裂过程及其致病性等方面的生物学特性,以确定两个β-微管蛋白基因功能的差异。研究结果表明,β1-微管蛋白基因敲除突变体菌株的生物学表型与野生菌株差异不显著；而敲除β2-微管蛋白基因的突变体菌株尽管也能完成其生活史,但其菌丝生长变慢、分支增多、无性及有性生殖能力下降、致病力降低。由此可以看出β2-微管蛋白基因对禾谷镰孢菌是必需的而β1-微管蛋白基因是非必需的,但是两者在功能上是可以相互替代的。同时用荧光蛋白基因标记这两个β-微管蛋白基因以测定其时空表达,结果显示禾谷镰孢菌β1、β2-微管蛋白基因在各时期的菌体细胞内均是同时表达的,并都参与微管的组装。该结果进一步说明两者在功能上是互补的。以上研究结果表明禾谷镰孢菌β1、β2-微管蛋白基因在各种菌体细胞内均是同时表达的,并都参与微管的组装；两者在生物学功能上有些差异,但是在功能上互补。5禾谷镰孢菌对多菌灵抗性的分子机理大多数植物病原真菌苯并咪唑杀菌剂抗性的产生是由于β-微管蛋白基因发生突变。禾谷镰孢菌具有两个β-微管蛋白基因,前期研究表明其多菌灵抗性菌株β1-微管蛋白基因未发生突变,而仅β2-微管蛋白基因发生突变,其突变主要发生在编码167、198和200位氨基酸的碱基上。为了进一步证明β2-微管蛋白基因突变与多菌灵抗性的关系,本研究选取一株禾谷镰孢菌田间多菌灵高抗菌株,分别敲除其β1、β2-微管蛋白基因,测定各敲除突变体菌株的多菌灵敏感性,结果发现敲除β2-微管蛋白基因的突变体菌株对多菌灵超敏感,并且与野生菌株β2-微管蛋白基因敲除突变体菌株对多菌灵的敏感性一致；而敲除β1-微管蛋白基因的突变体菌株对多菌灵超高抗。Giemsa染色观察多菌灵处理这两类敲除突变体对其有丝分裂的影响,结果也证实了它们对多菌灵敏感性的变化。应用绿色荧光蛋白GFP标记同一田间抗性菌株的β1、β2-微管蛋白,荧光显微观察多菌灵的作用,结果进一步直接证实了该菌株β2-微管蛋白对多菌灵敏感性的降低。同时分别用禾谷镰孢菌多菌灵田间抗性的4种突变方式的β2-微管蛋白基因来恢复野生菌株的β2-微管蛋白基因敲除突变体菌株,获得的各类型恢复突变体菌株对多菌灵的敏感性与其恢复突变β2-微管蛋白基因DNA片段来源菌株对多菌灵的敏感性一致。以上研究结果表明禾谷镰孢菌田间多菌灵抗性是由于β2-微管蛋白基因的突变引起的,该基因的不同的突变方式导致对多菌灵抗性的差异。同时还表明禾谷镰孢菌多菌灵抗性突变的β2-微管蛋白基因可以用作禾谷镰孢菌遗传转化的显性筛选标记。更多还原

【Abstract】 Microtubules, as steady-state dynamic polymers composed ofα-andβ-tubulin, are ubiquitous eukaryotic cell structures that are involved in a variety of intracellular processes including morphogenesis, cell growth and division. Beta-tubulin is the target of benzimidazoles, and the resistance of these fungicides is associated with amino acid substitutions at special codons ofβ-tubulin. Fusarium graminearum is the primary pathogen causing Fusarium head blight of wheat in China, and carbendazim (methyl benzimidazol-2-yl carbendazim, MBC) is one of the key fungicides used to control this disease, but the action and resistance phenotypes of this agent are different for F. graminearum than for most other filamentous pathogenic fungi.The purposes of this dissertation are to (1) understand the action mechanism of carbendazim on F. graminearum by compare the conidial germination and mitosis in germlings of carbendazim-sensitive and-resistant strains of F. graminearum and B. cinerea; (2) determine the target of carbendazim in F. graminearum by deletion theβ1-orβ2-tubulin gene and microtubules labeled by expressing the GFP taggedβ1-orβ2-tubulin; (3) study the physiological functions of the two P-tubulin genes in F. graminearum by compare the characteristics of mutants deletedβ1-orβ2-tubulin gene; (4) analysis the expression of the two P-tubulin genes in F. graminearum by taggingβ1-andβ2-tubulin with GFP and RFP; (5) confirm the carbendazim resistance mechanism by construction the putative carbendazim-resistantβ2-tubulin gene-deletion and-complementation mutants of F. graminearum. These results provide a theoretical foundation to control Fusarium head blight of wheat and manage carbendazim-resistance in F. graminearum.1 Mitotic Division in Germ Tube of Fusarium graminearumThe nuclear phase and mitosis in germ tube of Fusarium graminearum were studied with staining by Giemsa techniques. There was single nucleus in conidial cell. After division in conidial cell, the nuclei entered into germ tube and divided repeatedly, and therefore the number of nuclei in germ tube variably multiplied. The mitosis of F. graminearum has four phases. The chromosomes gradually shortened and thickened during prophase, and at metaphase the chromosomes were distinctly visible. The chromatids separated at anaphase and moved to opposite poles, and then, daughter nuclei were shaped at telophase. Synchronous and asynchronous chromosome separation was seen, and it was more common that the lagging chromosomes formed anaphase bridges.2 Effects of Carbendazim on Conidial Germination and Mitosis in Germlings of Fusarium graminearumPrevious data indicated that the action mechanism of benzimidazoles on most filamentous pathogenic fungi, e.g. Botrytis cinerea, is inhibition mitotic division, and resistance to benzimidazoles has been associated with mutations in theβ-tubulin gene. The effects and the different resistance mechanism of carbendazim in Fusarium graminearum was found, by comparison of the effects of carbendazim on conidial germination and mitosis in germlings of F. graminearum and B. cinerea. Conidia and germlings of F. graminearum wild type strains, as B. cinerea, germinated or grew in the presence of carbendazim to produce distorted, more branched, and elongation-restrained germlings and more branched foreparts. And in carbendazim-treated germlings there were irregularly distribution of chromosomes masses and no normal nuclei division was able to be observed. The CMIs (chromosome mitosis index) rapidly increased within 60 min and after that rapidly dropped. Compairing the carbendazim-resistants, however, F. graminearum was different from B. cinerea in growth and mitosis when they were treated with carbendazim. F. graminearum carbendazim-resistant strains, as ones of their wild-type, they produced significantly more branches and the CMIs have significant variation although abnormal mitosis was not observed under treatment of carbendazim. While B. cinerea carbendazim-resistant strains expressed normally in morphology of growth and mitosis after carbendazim treatment. The results show that carbendazim inhibits mitosis in F. graminearum and there should be a novel mechanism for carbendazim resistance in F. graminearum.3 Target of Carbendazim on Fusarium graminearumIn this study, we deleted theβ1-andβ2-tubulin gene of F. graminearum, respectively, and measure the sensitivity of carbendazim on deletion mutants. The results indicated that theβ1-orβ2-tubulin gene-deletion mutants are carbendazim-sensitive, but there different sensitivity in these mutants to carbendazim. The same results have been obtained by acquirement the mutants which microtubules labeled by expression the GFP taggedβ1-or P2-tubulin, and determination the carbendazim-binding affinity onβ1-orβ2-tubulin of F. graminearum by observation the characteristics of microtubules in these mutants under carbendazim treatment. The results show that the affinities are different forβ1-tubulin than forβ2-tubulin.The present results indicate that bothβ1-andβ2-tubulin of F. graminearum are the targets of carbendazim, and there is different carbendazim-binding affinity of the twoβ-tubulins. This difference may be associated with amino acid diversity at codon 240 ofβ-tubulin. The present work also reveals clearly that bothβ1-and P2-tubulin of F. graminearum, tagged with GFP and replaced one or two endogenous P-tubulins with a tagged version of the same gene, is apparently biologically active, and expression does not impact cell physiology.4 Functions of Twoβ-Tubulin Genes in Fusarium graminearumThe biological characteristics of theβ1-andβ2-tubulin gene deletion mutants of F. graminearum were assayed, including vegetative growth, asexual and sexual reproduction, mitosis and pathogenicity, to detect the physiological functions of the two P-tubulin genes. The results indicate that there are no significant different biological phenotypes inβ1-tubulin gene deletion mutants from wild type strains. Although they are active and can complete their life cycles, P2-tubulin gene deletion mutants have slow growth, more branches, decreased asexual and sexual reproduction ability, and poor pathogenicity. All mutants exhibit normal mitosis. These data indicate thatβ2-tubulin gene is more essential for activity thanβ1-tubulin gene, and there may be some functional differences between them, but the two P-tubulins are functionally interchangeable.The same results have been obtained by acquirement the mutants which microtubules labeled by expression the GFP taggedβ1-orβ2-tubulin, and determination the carbendazim-binding affinity onβ1- orβ2-tubulin of F. graminearum by observation the characteristics of microtubules in these mutants under carbendazim treatment.We constructed gene fusion mutants whichβ1-or/andβ2-tubulin was/were replaced by a GFP/RFP tagged version of the same gene, and determined temporal and spatial expression of the two P-tubulin genes. These data indicate that the two highly divergent P-tubulin genes in F. graminearum are both constitutively expressed during all lifecycle stages in all cells and individual microtubules can be composed of combinations between these different isotypes.All these data demonstrate that the twoβ-tubulins are substantially functionally interchangeable, but suggest that there may be subtle functional differences between them. 5 Molecular Mechanisms of Carbendazim Resistance in Fusarium graminearumBenzimidazole fungicides are thought to act by bindingβ-tubulin and preventing its polymerisation to microtubules. Resistance is believed to be associated with mutations in theβ-tubulin gene resulting in altered binding affinity in most filamentous pathogenic fungi. Previous studies have indicated that two P-tubulin structural genes in F. graminearum, and carbendazim-resistance is not due toβ1-tubulin but due toβ2-tubulin mutation included at codon 167,198 and 200. More evidence is required to confirm this carbendazim-resistance mechanism in F. graminearum.In this study, we deleted theβ1-andβ2-tubulin gene of field carbendazim-resistant strain of F. graminearum, respectively, and measure the sensitivity of carbendazim on deletion mutants. The results indicated that theβ2-tubulin gene-deletion mutants are super-sensitive to carbendazim, same as the mutant deleted theP2-tubulin gene of wild type strain. But theβ1-tubulin gene-deletion mutants are very high resistant to carbendazim, namely, more resistant to carbendazim than the field carbendazim-resistant strain whichβ1-tubulin gene was deleted. These results are confirmed by observation the effects of carbendazim on mitosis of these mutants through Giemsa-stain method. We obtained the mutants which microtubules labeled by expression the GFP taggedβ1-orβ2-tubulin of the field carbendazim-resistant strain, and determination the carbendazim-binding affinity onβ1-orβ2-tubulin by observation the characteristics of microtubules in these mutants under carbendazim treatment. The results further directly verify that the carbendazim-binding affinity onβ2-tubulin of this field resistant strain is significantly lower than wild type strain. Four kinds of amino acid substitutions inβ2-tubulin gene of carbendazim field resistant strains were used to replace the P2-tubulin gene of wild type strain. All mutants are resistant to carbendazim, and the resistance level of these mutants is consistent with these field resistant strains.Altogether these observations confirm that carbendazim-resistance in F. graminearum is due toβ2-tubulin gene mutation, different amino acid substitutions inβ2-tubulin are correlated with different sensitivity to crabendazim andβ2-tubulin gene of carbendazim-resistant strains can be used as a dominant selectable marker in F. graminearum transformation.更多还原