【作者】 赵伟；

【导师】 王源超；

【作者基本信息】 南京农业大学 ， 植物病理学， 2010， 博士

【摘要】 大豆是世界上重要经济农作物,大豆疫霉(Phytophthora sojae)侵染大豆而引起幼苗猝死和成株的根腐病是世界大豆生产上的主要病害之一,每年给世界的大豆产业带来巨额经济损失。大豆疫霉通过感受性激素、外界温度、湿度及条件的变化而进入不同的生活周期,在侵染寄主的过程中通过感知寄主而分泌大量的降解酶、坏死蛋白、蛋白酶抑制剂等效应因子以达到成功侵染寄主的目的,因此研究大豆疫霉如何感受外界的信息及信号传导机制、效应因子的分泌机制可以为防止病害的发生和扩展提供理论依据。但由于传统的、在其他生物中运用成熟的基因敲除等分子遗传操作技术不能运用在大豆疫霉中,因此对大豆疫霉的生长发育规律及致病性机理了解的相对较少。随着大豆疫霉全基因组数据、转录组数据的不断公布及完善,开发一种快捷、易操作、高通量的基因功能研究技术平台就显得尤为重要。大豆疫霉中利用双链RNA(dsRNA)介导的基因沉默。dsRNA介导的转录后基因沉默在真核生物中是普遍存在并且非常保守的一种降解靶标基因的机制。本研究将体外合成的dsRNA通过PEG介导的原生质转化方法转入大豆疫霉中从而介导目的基因的瞬时沉默,该技术不仅能利用单个dsRNA介导单个基因的沉默,更能够将不同基因合成的dsRNA同时介导多个内源基因的同时沉默。在本研究中选择了两个目的基因作为研究的靶基因,分别为细胞循环调控基因PsCdc14(调控有丝分裂细胞循环的蛋白质磷酸化酶)和一个无毒基因PsAvr3a(能被大豆抗病基因Rps 3a所识别的RxLR效应因子)。将体外分别合成的PsCdc14和PsAvr3a dsRNA转入大豆疫霉原生质体后,再生的转化子到第8天能够观察到转化子内源靶基因mRNA的减少；第9天到15天都表现出显著的基因沉默现象,沉默效率在50%-95%之间；第17天,所有的沉默转化子中内源基因都恢复到正常转录水平。本研究针对PsCdc14和PsAvr3a的基因沉默均导致了良好的表现型,证明dsRNA介导的大豆疫霉基因瞬时沉默系统可以用来鉴定基因功能,其耗时短、效率高、高通量操作的特点加快大豆疫霉功能基因组研究进展。大豆疫霉G蛋白偶联受体候选编码基因的生物信息学预测及转录分析。G蛋白偶联受体(G protein coupled receptors,GPCR)是真核生物中普遍存在的一类重要跨膜受体蛋白家族,通过感受外界信息后将信号传导到细胞内,从而激活细胞内的一系列信号传导途径,并最终影响生物个体的生长发育。本研究通过生物信息学方法方法从大豆疫霉、橡树疫霉和致病疫霉的全基因组数据库中挖掘出大量GPCR候选编码基因。对三种疫霉中GPCR候选基因之间进行了同源性比较,分析了基因在基因组中的结构特征。利用RNA-sequencing和qRT-PCR等技术分析了大豆疫霉中GPCR候选编码基因在生活史及侵染各个阶段的转录情况,为后续功能基因研究提供了思路，为大豆疫霉防治及新的药物靶标筛选提供了理论基础。新型G蛋白偶联受体PsPIPK4和PsPIPK10分别参与调控大豆疫霉的无性发育及有性生殖。游动孢子具有趋化性的特点,通过感受植物表面产生的物理或化学信号来后接近寄主、休止、萌发从而成功侵染寄主。大豆疫霉有性生殖产生具有厚壁的卵孢子,可以抵抗外界的不良环境从而比营养繁殖体存活的更久,同时卵孢子也是病害主要传播手段,是病害流行的初侵染源。生物信息学分析发现大豆疫霉的基因组中含有12个新型G蛋白偶联受体,GPCR-PIPK家族,其C端为GPCR所具有的典型的7次跨膜结构,而在N端具有磷脂酸肌醇激酶(PIPK)的结构域。qRT-PCR结果显示其中的一个编码基因PsPIPK4在休止孢及休止孢萌发阶段上调表达。功能分析证明PsPIPK4沉默突变体游动孢子的游动速率变慢,并且很快休止,导致游动孢子的趋化性丧失；休止孢的萌发显著降低,导致游动孢子的致病性减弱。另一个编码基因PsPIPK10在无性发育及侵染阶段都具有较高的转录量。功能分析证明PsPIPK10沉默突变体卵孢子产量急剧减少,所产的卵孢子量只有野生型菌株的1-5%。这也是在卵菌中第一次报道候选的GPCR编码基因参与调控无性发育及有性生殖。大豆疫霉SNARE蛋白家族生物信息学预测及PsYKT6的功能分析。在真核生物中,膜泡运输参与了细胞体内一系列的生理生化反应,这其中就包括调节细胞的极性生长及一些外泌蛋白的分泌。SNARE(可溶性N-乙基马来酰胺敏感因子附着蛋白膜受体)蛋白是真核生物细胞膜泡运输过程中发生膜融合最重要的调控蛋白家族,在真核生物中非常保守。在本研究中,通过生物信息学方法在大豆疫霉基因组中预测到35个编码SNARE蛋白的基因。利用转录谱RNA-sequencing测序技术对这35个基因在无性发育及侵染阶段进行了转录分析,发现大多数基因在发育和侵染的各个阶段都具有较高的转录水平,推测其可能在大豆疫霉的生长发育及侵染过程中具有重要作用。进一步利用疫霉菌稳定遗传转化技术将其中最为保守的一个SNARE基因PsYKT6沉默后的结果证实,PsYKT6在大豆疫霉的有性生殖、无性发育及致病性方面都发挥着重要调控作用。更多还原

【Abstract】 Phytophthora sojae is the casual agent of damping off of the seedlings and root rot disease of soybean, which is an economically important crop around the world. It is one of the most notorious pathogens which caused tremendous economic loss in soybean production annually. P. sojae switch the life stages by sensing hormone, temperature, humidity and other environmental conditions.During infection of their hosts, P. sojae secreted a variety of extracellular proteins to promote the pathogenicity. Therefore, to study the mechanism that P. sojae sense the environmental factors and the signal transduction pathway, and the process that P. sojae secretie extracellular proteins will provide theoretical bases for disease control. Methods for reverse genetics involving gene disruption are not currently feasible in oomycetes due to low rates of homologous recombination during transformation and diploidy. The molecular mechanisms underlying development and infection are largely unknown. As the increasing mount of the genome sequence and transcription profile data for P. sojae available, a feasible and efficient genetics protocols is critical for studying the biology, pathogenicity, and evolution of these organisms.Double-stranded RNA mediated gene silencing in Phytophthora sojae. Double-stranded (ds)RNA mediated gene silencing, a post-transcriptional and highly conserved process in eukaryotes, results in specific gene silencing through degradation of the target mRNA. Recently, we set up a dsRNA mediated transient gene silencing protocol combined with in vitro dsRNA synthesis and a polyethylene glycol (PEG) mediated transformation system. Single-gene silencing is accomplished by introducing unique dsRNA fragments into protoplasts, and multiple-gene silencing is performed with chimeric overlapping dsRNA fragments. The cell cycle regulated gene PsCdcl4 (a protein phosphatase that regulates the mitosis and the cell cycle) and the avirulence gene PsAvr3a (an RxLR effector that is recognized by the soybean Rps3a resistance proteins) are transiently silenced by introducing in vitro synthesized dsRNA into P. sojae protoplasts. The results showed that there is no reduction in PsCdc14 or PsAvr3a mRNA in the transformants until 8 days after the specific gene dsRNA was transferred into protoplasts. From 9 to 15 days, the transformants exhibited significant gene silencing, with mRNA levels reduced by 50% to 95%. All the silencing transformants showed recovery of gene expression by 17 days. Thus, the gene silencing protocols could significantly reduce the mRNA level of PsCdcl4 and PsAvr3a, yielding detectable phenotypes. The successful setup of transient gene silencing could make P. sojae high-throughput gene function elucidation work feasible, facilitating our understanding of the development and pathogenicity mechanisms of this organism.Bioinformatics prediction and transcription analysis of GPCR candidate coding gene in phytophthora sojae.G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors in eukaryote and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks, affecting the development and metabolic pathway. Phytophthora sojae changes the life stages by sensed the hormone, temperature, humidity and other environmental condition. Consequently, to study the mechanism of P. sojae sense the environmental information and the signal transduction pathway will provide theoretical bases for disease control. In this study, we predicted many putative GPCRs coding genes in the genome of P. sojae, P. ramorum and P. infestans according to the sequences and domains from reported GPCR in other organisms. The ortholog alleles and the gene genomic distribution patterns of the putative GPCR genes of P. sojae, P. ramorum and P. infestans were analyzed. In P. sojae, the transcription profile of putative GPCR was also performed by RNA-sequencing and qRT-PCR. These results provide a new study way for analysis function gene, which is crucial to disease control and drug development.Two novel protens, PsPIPK4 and PsPIPK10 are respectively involved in asexual development and sexual reproduction in Phytophthora sojae. P. sojae, zoospores have the characteristic of chemotaxis, which can swim chemotactically toward compounds released by roots of their host plants, and then encyst on the root surface from where the hyphae penetrate the root directly from the cyst. Sexual oospores are often thick walled and thus more durable than vegetative structures. Also, sexual oospores are the main agents of dispersal for P. sojae. The genome sequencing of P. sojae provide a great opportunity to identify a novel group of GPCRs, termed as GPCR-PIPKs, which fused a typical GPCR 7-transmembrane domain at the N-terminusl combined with a phosphatidylinositol phosphate kinase (PIPK) domain at the C-terminus. PsPIPK4, one of GPCR-PIPks, was upregualted in cyst and cyst germination. The function analysis indicated zoospore encystment and cyst germination were badly affected, resulting in the inability of chemotaxis and infecting soybean in PsPIPK4-silenced mutants. PsPIPK10, another GPCR-PIPks, was constitutive expression in asexual development and infection stages. PsPIPK10-silenced mutants exhibit remarkable reduction of oospore number, just around 1-5% production compared to the wild type. This is the first reported that putative GPCRs are involved in asexual development and sexual reproduction according to our current knomledge.Genome-wide identification and transcriptional profiling of SNARE family genes and functional characterization of PsYKT6 in Phytophthora sojae. In eukaryotic cells, the intracellular vesicle trafficking is essential for many aspects of cellular processes including polarized growth and secretion of extracellular proteins. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Here, we identified 35 genes, which might be a total set, encoding putative SNARE proteins that are key factors for vesicular trafficking, taking advantage of available whole genome sequence in the oomycete plant pathogen Phytophthora sojae. The subsequent systematic analysis to determine its transcription in different development and infection stages revealed that most putative SNAREs show important roles in P. sojae. One of the most conserved SNARE proteins (PsYKT6) was functionally characterized by homology-dependent gene silencing method. The phenotype analysis showed that PsYKT6 plays key roles in both asexual development and sexual reproduction, and in pathogenesis on host soybean cultivars.更多还原