【作者】 王秦虎；

【导师】 单卫星；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 病原菌严重影响植物的生存、生长和生殖等,危害巨大。植物病原卵菌可造成毁灭性的作物生产损失,威胁自然生态系统。卵菌为二倍体真核微生物,其形态和生理上与丝状真菌相似,但在进化上与硅藻和褐藻更近,多数杀真菌剂对卵菌病害的防控无效。卵菌包括疫霉菌、霜霉菌、腐霉菌和白锈菌等。引起爱尔兰大饥荒的致病疫霉菌,每年仍可造成多达67亿美元的经济损失。引起大豆幼苗倒伏或大豆根腐病的大豆疫霉菌,每年造成约10-20亿美元的经济损失。作物病害最有效的防控依赖于抗病性的利用,然而病菌的遗传变异,包括可能的表观遗传变异,频频引起作物抗病性丧失。许多研究者多次观察到疫霉菌的表观遗传现象。小RNA分子,例如si RNA,mi RNA和pi RNA,可通过反义互补配对引起转录水平和转录后水平基因沉默,是真核生物重要的表观遗传调控分子。然而,我们对疫霉菌的小RNA的种类、特征、功能和作用方式等知之甚少。为此,我们以大豆疫霉菌为模式,对其阶段发育和毒性变异相关的小RNA进行分析,主要研究结果如下:1、通过深度测序、计算机识别、传统克隆和Northern杂交等方法,在大豆疫霉菌基因组中发现了大量ts RNA分子(t RNA来源的小RNA分子)。利用自主开发的软件ts RFinder对大豆疫霉菌营养菌丝小RNA的重测序和Northern杂交分析结果表明,t RNA reads富集在29-36 nt,最高峰在33 nt,其5’第一个碱基偏好于碱基G,其次为T。在28-45 nt的小RNA中,t RNA reads高达12.34%,丰度最高的ts RNA-Gly CCC-5p占总reads的2.05%。2、基因组分析和Northern杂交分析表明,ts RNA是一类保守的小RNA分子。Northern杂交分析还表明,其表达模式也相对保守:大多数ts RNA在卵孢子、营养菌丝和孢子囊时期高表达,在侵染和萌发的休止孢时期低表达,而在休止孢时期几乎不表达。通过生物信息分析,我们预测到多个ts RNA候选靶标基因。数字基因表达谱、实时定量反转录PCR等结果表明,大部分候选靶标基因表达模式与ts RNA的表达模式负相关。因此,疫霉菌ts RNA分子可调控靶标基因的表达。3、利用RNA定量和RLM-RACE技术(RNA连接介导的c DNA末端快速扩增技术),确认并捕获到5个靶标基因在大豆疫霉菌菌丝中的降解产物。然而,这些ts RNA靶标基因降解位点不在小RNA的结合位点,与已知的mi RNA介导的靶基因降解机制差异较大。与RLM-RACE结果一致,高通量RNA末端并行测序表明,ts RNA可介导靶基因在邻近结合位点的位置降解,略偏好于其结合位点下游区域。疫霉菌瞬时转化分析表明,疫霉菌ts RNA可序列特异的抑制GUS人工靶标基因的表达。因此,疫霉菌ts RNA分子可与靶标基因反义匹配并引起m RNA降解。4、通过对大豆疫霉菌效应基因Avr1b-1沉默菌株的小RNA深度测序和Northern杂交分析,发现两类与Avr1b-1同源的小RNA分子:即Avr1b-1沉默的触发子Avr1b-1-s RNA和沉默信号Avr1b-1-si RNAs。杂交结果还表明,这两类小RNA都在毒性菌株中累积,而在无毒菌株中不累积。其中,触发子Avr1b-1-s RNA比沉默信号Avr1b-1-si RNAs的累积早12-24小时。进一步的生物信息分析和Northern杂交分析表明,大豆疫霉菌中存在大量与RXLR效应蛋白基因同源的小RNA分子,并且RXLR基因的表达呈负相关。5、基因组分析和反转录PCR数据表明,Avr1b-1位点是双向转录的,其形成的天然反义转录本对为Avr1b-1-s RNA的前体,并通过其产物触发子Avr1b-1-s RNA来调控无毒基因Avr1b-1的表达。反转录PCR数据还表明,Avr1b-1反义链Avr1b-1(-)的差异表达对触发子Avr1b-1-s RNA在毒性菌株和无毒菌株间的差异累积负责。进一步的序列分析表明,Avr1b-1(+)和Avr1b-1(-)的表达水平与其启动子中10 nt左右INDEL(插入和缺失变异)的存在和缺失相关联。更多还原

【Abstract】 Pathogens cause severe damages to the survival, growth and reproduction of plant. The pathogenic oomycetes are destructive to crop production and threaten natural ecosystems. Oomycete are diploid eukaryotic microorganisms, which are similar to filamentous fungi in morphology but close to brown algae and diatoms in evolution, making fungicides ineffective against oomycetes. Well-known oomycetes include Phytophthora, Peronospora, Pythium and Albugo. The causal agent of Irish potato famine, Phytophthora infestans, causes $6.7 billion potato losses annually. Phytophthora sojae causes damping-off of seedlings and root rot of soybean, with an annual cost of $1-2 billion worldwide.Use of host resistance is the most effective approach for crop disease control. However, genetic variation of pathogens, including possible epigenetic variations that were observed in the genetic variation of Phytophthora, causes frequent crop resistance failure. Small RNAs, such as si RNA, mi RNA and pi RNA, are able to pair complementarily with its antisense transcript locally and lead to transcriptional and posttranscriptional gene silencing. They are important molecules in epigenetical regulation but little is known on diversity, characteristics, functions and underlying regulatory mechanisms of Phytophthora small RNAs. Therefore, we use P. sojae as a model to study the small RNA involved in the pathogen development and virulence variation, and the main findings are as follows:1. By using deep sequencing, computational identification, conventional cloning and Northern hybridization, we identified a large amount of ts RNAs(t RNA-derived small RNA) encoded by the P. sojae genome. Using algorithm developed for ts RNA identification and annotation, we further analyzed the small RNA re-sequencing data for the vegetative hyphae stage of P. sojae. The results are consistent with Northern blotting, being the t RNA reads enriched in 29-36 nt and peaked at 33 nt. The first nucleotide of t RNA reads is predominately G, followed by T. In the dataset within 28- 45 nt, the t RNA reads constitute to 12.34 % of the total reads, while the most abundant ts RNA-Gly CCC-5p comprises 2.05 % of the total reads.2. Genome analysis and Northern hybridization showed that ts RNA is a class of conserved small RNAs. Northern analysis also showed that the expression patterns of ts RNA are relatively conserved, being highly accumulated in oospores, mycelia and sporangia, and much lower in the infection stage and germinated cysts, while nearly not detected in cysts. Bioinformatics analyses showed a large set of potential ts RNA target candidates. Digital gene expression profiling and real-time quantitative RT-PCR results indicated that the expression levels of the most target genes are negatively correlated with accumulation levels ts RNAs, suggesting that ts RNAs are functional and may down-regulate gene expression in P. sojae.3. By using RNA quantification and RLM-RACE technologies(RNA ligation mediated-rapid amplification of c DNA ends), we confirmed degradation of five target genes in P. sojae mycelia. However, the degradation of target transcripts was not in the ts RNA binding sites. This is distinct from mi RNA-mediated degradation of target transcripts. Consistent with the RLM-RACE experiments, high-throughput PARE(parallel analysis of RNA ends) analysis showed that ts RNA-mediated degradation of target transcripts occurs near but not at the binding sites, being slightly in favor of the downstream region. Transient transformation analysis in P. sojae demonstrated that ts RNAs repress expression of artificial GUS targets, in a sequence-specific manner.4. By deep sequencing analysis of the effector gene Avr1b-1-silencing strain and Northern hybridization, two types of small RNAs homologous to Avr1b-1 were identified, the potential silencing trigger Avr1b-1-s RNA and the signal molecules Avr1b-1-si RNAs. Hybridization results also indicated that both types of small RNAs accumulated in the virulent but not in the avirulent strain. In addition, the trigger Avr1b-1-s RNA accumulated 12-24 hours earlier than the silencing signal Avr1b-1-si RNAs. Further bioinformatics analyses and Northern hybridization showed that vast small RNAs are associated with RXLR effector genes, and their expression levels were negatively correlated.5. Genome analysis and RT-PCR analysis indicated bidirectional transcription of Avr1b-1 and the natural antisense transcripts being the precursor of Avr1b-1-s RNA, the final product Avr1b-1-s RNA may regulates the expression level of Avr1b-1. The RT-PCR data also suggested that the differentially expressed Avr1b-1(-) is responsible for the differentially accumulated Avr1b-1-s RNA trigger in the virulent and avirulent strains. Further sequence analysis showed that the expression levels of Avr1b-1(+) and Avr1b-1(-) are likely associated with the presence or absence of the ~10 nt INDELs(insertions and deletions) in their promoter regions.更多还原