【作者】 王永林；

【导师】 王源超；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 大豆疫霉(Phytophthora sojae (Kaufman & Gerdman))已经成为一种非常重要的植物病原菌,由其侵染大豆引起的疫霉根腐病是大豆生产中的毁灭性病害之一。长期以来,由于大豆疫霉缺乏有效的遗传操作系统,对其生长发育和致病性分子机理的研究非常少。随着大豆疫霉全基因组测序的完成和遗传转化系统的逐步建立,大豆疫霉已经成为研究卵菌分子遗传的模式种。转录因子是基因表达调控的关键因子,从研究转录因子的功能入手来解析大豆疫霉生长发育与致病性过程中的分子机制,对了解疫霉菌发育的分子生物学和致病性具有重要意义,可以为寻找新的疫霉杀菌剂作用靶标,设计新的疫病控制策略提供理论依据。大豆疫霉C2H2锌指转录因子、热激转录因子(heat shock transcription factor)和MADS-box转录因子的生物信息学分析：转录因子在真菌生长发育和致病性方面有着重要的作用,对开展大豆疫霉转录因子的研究提供了有益的参考。由于对大豆疫霉生长发育和致病性机理缺乏研究,大豆疫霉全基因组测序的完成为全面地分析其转录因子带来了新的机遇。为了系统地研究大豆疫霉生长发育和致病过程中转录因子的功能,本文用已知真菌转录因子对大豆疫霉全基因组进行同源序列搜索,详细分析了C2H2锌指转录因子、热激转录因子和MADS-box转录因子这三类转录因子家族的结构特点和基因组分布情况。通过分析发现,大豆疫霉共有90个C2H2锌指转录因子,20个热激转录因子和1个MADS-box转录因子；C2H2锌指转录因子和热激转录因子在基因组上排列方式是丰富多样的,有随机分布的,也有几个基因成簇排列的。生物信息学分析为后续基因功能研究提供了依据。PsCZF1在大豆疫霉生长发育和致病过程中作用的研究：PsCZF1是一个编码C2H2锌指结构的转录因子,该基因在已测序的卵菌中是高度保守的。将PsCZF1的预测启动子与GUS报告基因融合后转化大豆疫霉得到稳定表达GUS的转化子,染色结果显示GUS的活性在卵孢子阶段最高,而且也受到侵染上调表达。为了详细研究PsCZF1的功能,本研究利用基因沉默技术,获得了该基因的沉默突变体。PsCZF1沉默突变体的生长速率与野生型相比下降了约50%,然而在菌丝细胞大小、孢子囊形态等方面与野生型菌株无显著差异。沉默PsCZF1后严重影响了卵孢子形成、游动孢子的产量和休止孢的萌发。接种结果显示PsCZF1沉默突变体降低了对大豆的致病性。结果表明了卵菌特异的C2H2锌指转录因子PsCZF1在大豆疫霉的生长发育和致病性中起着重要的作用。大豆疫霉热激转录因子PsHSFl参与应答氧化压力、热激胁迫和致病性：PsHSF1编码的蛋白质含有HSF1DNA结合域。生物信息学分析发现PsHSF1与其有一定同源性的基因成簇排列在基因组上,其他卵菌中找不到序列上高度同源的基因。利用RT-PCR分析PsHSF1在无性生活史阶段的表达情况,结果表明PsHSF1只在休止孢及其萌发阶段表达,而且在萌发阶段表达量显著上调。同时,在过氧化氢胁迫下该基因受诱导表达。本文利用大豆疫霉遗传转化系统和基因沉默技术,获得了PsHSF1基因沉默的突变体。PsHSF1基因沉默突变对过氧化氢和高温处理变得敏感,但是不影响菌丝的正常生长和孢子囊的产生。此外,PsHSF1的沉默显著降低了休止孢的萌发率。致病性测定结果表明,PsHSF1沉默突变体的致病性显著延缓。结果揭示了热激转录因子PsHSF1参与了大豆疫霉抗氧化作用和致病过程。大豆疫霉MADS-box转录因子的研究：PsMAD1编码大豆疫霉MADS-boxDNA结合域的基因,该基因在其他疫霉菌中高度保守,系统发育分析揭示了疫霉菌编码MADS-box的基因与动物肌细胞增强因子(myocyte enhancer factor, MEF)家族的亲缘关系更近。利用RT-PCR方法和GUS报告基因,本研究详细分析了PsMAD1在大豆疫霉生活史和侵染过程中的时空表达,结果显示PsMAD1在大豆疫霉无性生活史阶段是组成性表达的,但在产孢菌丝、休止孢和萌发的休止孢时期表达量明显提高,而且PsMAD1受侵染的诱导呈现上调表达趋势。对该基因启动子序列分析找到了两个类似TATA-box的序列和卵菌核心启动子序列。这些结果暗示着PsMAD1作为转录因子可能在大豆疫霉生活史和侵染中发挥着重要的作用。更多还原

【Abstract】 Phytophthora sojae (Kaufman & Gerdman), an Oomycete causing one of the most destructive diseases on soybean worldwide,has become an important agricultural pathogen. P. sojae has been developed as a model species for the genus, having in place excellent genetic and genomics resources (including genetic maps, BAC libraries,and EST sequences),being established gene-silencing for funcational study, as well as having a well organized community of researchers.Transcription factors are key proteins which regulate gene expression and are of great value for study. Studying the molecular mechanism of transcription factors underlyging growth, development, and pathogenicity of P. sojae will help research to find novel targets of chemical control and provide new sight and good idea for designing comprehensive management of Phytophthora disaeases.Bioinformatics analysis of C2H2 zinc finger transcription factors, heat shock transcription factors and MADS-box transcription factors in genome of Phytophthora soja:The completion of genome sequencing of P. sojae has provided novel insights and platform to find and study TFs.In order to comprehensively functional study of TFs during growth, development, and pathogenisis, we used known TFs protein sequence from fungi and plants as query sequences with BLAST algorithm to explore genome-wide inventory of three classes of TFs,such as C2H2 zinc finger transcription factors, heat shock transcription factors and MADS-box transcription factors, and analyze motif or domain organization and their distribution in genome in details.The results showed that some genes of the same family often clustered in genome.All these bioinformatics analysis provided a useful clue to characterize TFs in P. sojae.PsCZFl gene encoding a C2H2 zinc finger protein is required for growth, development and pathogenesis in Phytophthora sojae:C2H2 zinc finger proteins form one of the largest families of transcriptional regulators in eukaryotes.We identified a Phytophthora sojae C2H2 Zinc Finger (PsCZF1),which was highly conserved in the sequenced oomycete pathogens.In the transformants of P. sojae containing the PsCZF1 promoter fused to theβ-glucuronidase (GUS)reporter gene, GUS activity was highly induced in the P. sojae oospores stage and upregulated by the infection. To elucidate the function, expression of PsCZF1 was silenced by introducing antisense constructs into P sojae. PsCZFl silencing transformants exhibited none alternation in the cell size and morphology of sporangia and hyphae; however, the hyphal growth rate was around 50% reduced in the mutants. PsCZF1-deficient mutants were also impaired in the production of oospores,swimming zoospores and the germinating cysts, indicating that the gene was involved in the various stages of the life cycles.Furthermore, we found PsCZF1-deficient mutants completely lost the virulence on the host soybeans.Our results suggested that this oomycete specific C2H2-type zinc finger protein played the important roles in the growth, development and pathogenesis,therefore, PsCZF1 might be an attractive oomycete-specific target for chemical fungicides screening.A heat shock transcription factor PsHSFl is involved in oxidative stress, heat shock and pathogenicity in Phytophthora sojae:Analysis of heat shock transcription factors (HSF) in genome of Phytophthora sojae revealed one gene PsHSF1 encoding HSF, which was clustered together with other highly homologous HSF in P. sojae genome. Bioinformatics analysis showed that there hardly existed high orthologs in other oomycete pathogens.In this paper, RT-PCR was used to analyze expression of PsHSF1 during asexual life stage and oxidative stress mediated with hydrogen peroxidase.These results indicated that PsHSF1 is uniquely expressed in cysts and germinated cysts, and obviously upregulated in latter stage; PsHSF1 also was induced by oxidative stress mediated with hydrogen peroxidase. To elucidate its molecular function, expression of PsHSF1 was silenced by introducing antisense constructs into P. sojae.Silencing of PsHSF1 did not disturb hyphal growth or sporulating, but severely affected hyphal growth on media containing hydrogen peroxidase or inoculated at 30℃. Furthermore, PsHSF1-silenced mutants showed decreased germination ratio.Finally, the pathogenicity of PsHSFl-silenced mutants were depressed. All the results revealed that PsHSFs would play important roles in antioxidative function and pathogenicity in P. sojae.Study on MADS-box transcription factor of P. sojae:MADS-box transcription factors play important roles in reproductive and vegetative development, and in signal transduction. Previously, in silico exploration in genome of soybean root rot pathogen, P. sojae,a gene PsMADl encoding MADS-box transcription factor has been identified. At the same time, the orthologs of other Phytophthora were identified.Phylogenetic analysis revealed that Phytophthora MADS-box genes are divided into animal subgroup of myocyte enhancer factor clade.Using RT-PCR and GUS reporter gen, this study reveled that PsMAD1 constitutively expressed during life stage, highly upregulated in sporulating hyphae,cysts and germinated cysts, furthermore, it was induced by early infection. Finally, examination of promoter region of PsMADl and its Phytophthora orthologs showed that their promoters contain two TATA-like element and one conserved 19-nt core promoter structure of oomycetes.These results indicated that PsMAD1 may play important roles during asexual life cycles and infection as a putative transcription factor.更多还原