【作者】 郑武；

【导师】 王宗华；

【作者基本信息】 福建农林大学 ， 植物病理学， 2006， 博士

【摘要】 Rho族蛋白是Ras超家族最主要成员之一，已发现的有Rho、Rac、Cdc42三个亚家族近百种蛋白。大量研究结果表明，Rho族蛋白在调控与肌动蛋白骨架相关的信号转导途径，并参与MAP激酶的细胞信号转导过程，起着调节细胞极性、细胞运动、细胞增殖、细胞凋亡、基因转录、细胞周期、微管活力、细胞转化、囊状运输途径、从吞噬细胞的NADPH氧化酶至酵母中葡聚糖合成酶等一系列酶活性、恶性肿瘤细胞的浸润和转移等重要作用。在真菌中，几乎所有Rho族蛋白都被证实参与调控细胞极性生长，其还参与调控细胞壁合成及其完整性、胞外分泌、细胞分化等；在丝状真菌中其还参与调控侵染致病过程。 稻瘟病菌(Magnaporthe grisea)是研究丝状真菌的重要模式生物，也是引起水稻重要病害稻瘟病的病原菌。该菌侵染循环过程经历了分生孢子萌发、附着胞分化、侵入栓的形成以及侵染生长，这一过程涉及复杂的细胞分化、信号转导和极性生长。 数据库分析结果表明，稻瘟病菌基因组中有7个Rho族蛋白成员，且通过基因表达分析表明这7个Rho族蛋白在稻瘟病菌中均可表达，因而我们推测这些Rho族蛋白在稻瘟病菌生长发育以及侵染致病过程中可能起着极为关键的作用。本论文选择了具有代表性的两个Rho族蛋白成员(MgCdc42和MgRho3)，深入开展了功能分析。 MgrCdc42在稻瘟病菌分生孢子中表达最强，通过构建插入失活突变体、持续激活突变体、负显性失活突变体以及过量表达突变体，分析了MgCdc42在稻瘟病菌中的功能。研究结果表明，MgCdc42插入失活突变导致稻瘟病菌侵入栓形成受抑，从而引起致病性显著下降；产孢量减少，分生孢子形态异常，气生菌丝生长受抑，菌丝异常；在疏水性表面分生孢子萌发延迟，附着胞形成滞后、减少，且附着胞异常。而MgCdc42负显性突变的结果与MgCdc42插入失活突变结果十分相似，最终也导致了稻瘟病菌分生孢子、营养菌丝异常，附着胞形成受抑，并导致了侵染能力下降。MgCdc42持续激活突变体之分生孢子正常，但其菌丝末端分枝增多，附着胞形成减少且侵染能力也大为下降。与这些突变体不同的是，MgCdc42过量表达突变体与稻瘟病菌野生型菌株之表型及侵染致病能力差异不显著。这些结果表明MgCdc42调控着稻瘟病菌细胞分化、极性生长以及其侵染致病过程。 MgRho3在稻瘟病菌分生孢子萌发阶段表达最强，通过构建插入失活突变体以及过量表达突变体，其在稻瘟病菌中的功能也得以解析。研究结果表明，MgRho3插入失活突变体侵染致病能力也显著下降，但与MgCdc42插入失活导致稻瘟病菌侵入栓形成受抑，并引起致病性显著下降不同的是，似乎MgRho3插入失活导致稻瘟病菌附着胞形成严重受抑，而引起侵染力下降。MgRho3插入失活还促使稻瘟病菌分生孢子从倒梨形改变为长椭圆形，产孢量减少；分生孢子萌发延迟，附着胞形成滞后且降低；菌丝末端分枝增多。与MgCdc42过量表达突变体不同的是，MgRho3过量表达突变体侵染能力提高，其可能是MgRho3过量表达突变体分生孢子可产生双附着胞引起的。MgRho3过量表达更多还原

【Abstract】 The Rho-family proteins make up a major branch of the Ras superfamily of small GTPases. To date, more than 100 members of Rho-family proteins have been described, and they belong to Rho, Rac and Cdc42 subfamily. Rho GTPases has been fuelled by the realization that they regulate many other signal transduction pathways in addition to those linked to the actin cytoskeleton. They participate in the regulation of cell polarity, gene transcription, G1 cell cycle progression, microtubule dynamics, vesicular transport pathways and a variety of enzymatic activities ranging from an NADPH oxidase in phagocytes to a glucan synthase in yeast. In fungi, almost all the Rho GTPases are shown to be involved in the regulateon of polarity growth , the synthesis of cell wall, cell development and exocytosis; In filamentous fungi, Rho proteins also control the pathogenicity.The rice blast fungus, Magnaporthe grisea, infects many economically important cereal crops, particularly rice. The interaction of M. grisea and rice is taken as a model for the study of fungus-plant interaction.When the fungal conidia land on and attach to a suitable surface, such as a rice leaf, a genn tube arises. Subsequently, germ elongation ceases and the tip of the germ tube swells to form an appressorium surrounded by a thick melanized cell wall. Turgor pressure builds up inside the appressorium, forcing a penetration peg into the plant tissues; this is then followed by invasive growth of the fungus. Thus, the host invasion by the rice blast fungus involves a complex cell morphogenesis requiring dynamic changes in polarity in response to host factors.7 putative Rho GTPases were found on the M. grisea genome based on the bioinformation analysis. RT-PCR assay showed that all of the 7 Rho GTPases were expressed in M. grisea. The results reminded us that these 7 Rho GTPases may all play an important role in control of the growth, development and pathogenecity of M. grisea. In this paper, the functions of MgCdc42 and MgRho3, two of the 7 Rho GTPases were analysed.The highest expression level of MgCdc42 was observed in conidia, and the function of MgCdc42 was dissected by analysis of MgCdc42 knock-out, constitutive activated, dominant negative and overexpressed mutants. The results showed that the MgCdc42 deletion led to the deficiency of penetration pegs, which decreased the pathogenicity of M. griease. MgCdc42 knock out mutant was also decreased in the conidiation and aerial hypha, delayed on the germination and appressoria formation, and was developed into abnormal conidia and appressoria. The similar results were found from the dominant negative mutation of MgCdc42. Although the conidia of the constitutive activated mutant were not changed, the mycelia branched more frequently at the ends, and mutants were decreased in the apressoria formation and pathogenicity. While overexpression of MgCdc42 had no significant effect on更多还原