寄生疫霉菌小RNA多样性及其参与基因表达调控的探索研究
本文关键词：寄生疫霉菌小RNA多样性及其参与基因表达调控的探索研究 出处：《西北农林科技大学》2017年博士论文 论文类型：学位论文
【摘要】：卵菌是一类形态上与真菌相似,但进化上和动植物以及真菌关系较远的真核微生物。其中疫霉属(Phytophthora)卵菌的多个病原物在世界范围内引起毁灭性的植物病害,每年造成严重的经济损失。由于进化上的独特性,卵菌在生理生化和侵染机制上与真菌差异很大,导致许多对真菌有效的杀菌剂对卵菌无效。因此探索疫霉菌-植物互作机制在植物病理学研究和田间生产实践中都具有重要的意义。另外,抗病品种的使用在田间病害防控中发挥着重要的作用,但疫霉菌毒性变异迅速,常通过丢失、突变无毒基因来逃避寄主抗病基因的识别,造成对应抗病品种失效。然而一些毒性菌株中的无毒基因尚未发生任何突变但基因不表达,这说明表观遗传学调控可能参与调节寄生疫霉菌毒性变异。基于此,我们分析了寄生疫霉菌(P.parasitica)小RNA的特点以及其在沉默内源基因尤其是致病相关基因方面发挥的功能。同时,为了了解疫霉菌-植物互作机制,我们围绕寄生疫霉菌-本氏烟模式互作体系,从基因组学和转录组学角度分析寄生疫霉菌与寄主植物亲和互作的分子机制。主要研究结果如下:1.通过高通量测序技术对寄生疫霉菌小RNA多样性的研究发现:(a)寄生疫霉菌累积大量的25-26nt和少量21nt小RNA,同时也累积一系列侵染阶段高度诱导表达的29nt和33nt的tRNA来源的小RNA。25-26nt小RNA和21nt小RNA具有强烈的5'碱基偏好性,无链特异性地成簇分布在基因组上。(b)超过7000个内源基因位点累积25-26nt小RNA,转录组测序表明这些基因一般在营养生长阶段和侵染阶段均不表达,qPCR实验支持转录组测序的结果。小RNA在外显子、内含子以及基因上下游区域均匀累积,说明基因沉默可能发生在转录水平。(c)累积25-26nt小RNA的基因主要为寄生疫霉菌特有的基因,包含大约40%的RXLR效应基因、50%的Crinkler效应基因和一些激发子基因,说明25-26nt小RNA在新基因沉默和寄生疫霉菌毒性变异中可能发挥重要作用。(d)25-26nt小RNA主要来源于基因稀疏区和基因密集区,且小RNA的产生和沉默状态能从重复区向两侧传播几百碱基到上千碱基距离。与此呼应,累积小RNA的内源基因大部分位于或十分邻近重复区域。(e)21nt小RNA主要来源于高表达基因的外显子区,其累积不足以导致基因完全沉默,且小RNA的产生可能依赖基因表达。(f)对已公布的致病疫霉菌的小RNA和转录组数据重新分析的结果表明,致病疫霉菌中25-26nt小RNA,而不是21nt小RNA,也与内源基因的沉默密切相关,这说明这一现象在疫霉菌中是保守的。2.通过对寄生疫霉菌侵染本氏烟(Nicotiana benthamiana)离体叶片不同时间点(0、3hpi、6hpi、12hpi、24hpi、48hpi)的样品进行转录组测序和分析,结果表明(a)侵染阶段早期和晚期基因表达差异很大,大量致病相关基因在侵染阶段早期上调表达而在晚期下调表达。基于此,我们鉴定了 231个侵染阶段早期特异性高表达的编码分泌蛋白的基因,这包括大量编码RXLR效应蛋白、NPP样激发子以及各种水解酶和蛋白酶抑制剂的基因。同时还发现编码盐酸小檗碱样蛋白、碳酸酐酶和SCP样胞外蛋白的三个家族基因以及多个功能未知的基因家族在侵染阶段早期特异性高表达。我们围绕其中一个家族进行系统地分析,发现其家族成员在疫霉中频繁复制且出现显著的结构域重排现象,根据结构域排列特点可将其分成三个亚家族。(b)大量蛋白翻译、核小体组装以及RNA甲基化相关基因在侵染早期显著上调表达,而三羧酸循环和ATP合成相关基因在侵染中后期上调表达。但与预期不符的是,囊泡介导的转运、微管介导的细胞运输、蛋白降解、蛋白磷酸化和信号传导相关基因在侵染阶段显著下调表达。3.此外,我们利用转录组数据对本氏烟基因转录本进行了组装和表达分析,并且通过鉴定抗病相关的基因来探索寄主植物防御疫霉菌的分子机制。结果表明(a)包括活性氧进发和程序化细胞坏死相关基因和PAMP诱导的基因在内的大量防御反应基因在侵染阶段上调表达。水杨酸、茉莉酸和乙烯的合成和信号通路相关基因均上调表达,进一步分析表明这些激素相关基因以及flg22/e1f18诱导表达的基因绝大部分在侵染阶段上调表达,且在3hpi和24hpi出现两个表达高峰,这可能是大部分抗病相关基因的重要特征。基于此,我们鉴定到了 3766个在3hpi和24hpi出现两个表达高峰的基因作为候选的抗病相关基因,其中753个在3hpi上调超过10倍。(b)系统地鉴定了本氏烟中的蛋白激酶和转录因子,它们大部分也在3hpi和24hpi出现两个表达高峰。我们进一步鉴定到了在3hpi和24hpi出现两个表达高峰的一系列受体样蛋白激酶和4个受体样蛋白,为PAMP/激发子受体筛选提供了重要的候选。(d)大量蛋白分泌、蛋白修饰以及包括糖异生、脂肪酸氧化以及三羧酸循环在内的分解代谢过程相关基因在侵染阶段显著上调表达;而以蛋白翻译、脂肪酸和糖类合成为代表的大量生物合成相关通路、光合作用以及离子转运等基础代谢过程相关基因在侵染阶段显著下调表达。
[Abstract]:The egg is a kind of form of bacteria and fungi are similar, but the evolution of eukaryotic microorganisms and plants and animals and fungi. Among distantly related Phytophthora (Phytophthora) multiple pathogens caused by the oomycete plant disease of destruction in the world, causing serious economic losses every year. By the unique evolution the differences in physiological and biochemical and fungal infection mechanism of large egg bacteria, causing many fungi on the sterilization of effective agent against oomycete Phytophthora is invalid. Therefore exploring plant interaction mechanism has important significance in the research and production practice in the field of plant pathology. In addition, the use of resistant varieties of play an important role in the field of disease prevention and control, but the toxicity of Phytophthora mutate rapidly, often through the loss of avirulence gene mutations to evade the host recognition of resistance genes and resistant varieties. However, failure caused by the corresponding toxicity in some strains of non-toxic base Because there is not any mutation but the genes are not expressed, indicating that epigenetic regulation may be involved in the regulation of Virulence Variation of Phytophthora parasitica. Based on this, we analyzed the parasitic Phytophthora (P.parasitica) characteristics of RNA and its endogenous gene in silence especially pathogenic related gene function. At the same time, in order to understand the interaction the mechanism of Phytophthora plants, we focus on Phytophthora parasitica - benthamiana mode interaction system, from the molecular mechanism analysis of Phytophthora parasitica and host plant compatible interaction point of genomics and transcriptomics. The main results are as follows: 1. by high-throughput sequencing of Phytophthora parasitica RNA diversity research: (a) Phytophthora parasitica accumulate a large amount of 25-26nt and a little 21nt RNA, but also accumulated a series of infection stage height to induce the expression of 29nt and 33nt from tRNA and 21nt small RNA.25-26nt small RNA RNA has a small 5'base strong preference, no chain specifically clustered in genome. (b) more than 7000 endogenous gene loci and accumulation of 25-26nt small RNA transcriptome sequencing showed that these genes generally in the vegetative growth stage and infection stage were not expressed and qPCR experimental support for transcriptome sequencing of small RNA results. In exon, Chi Ko and gene on the downstream area of uniform accumulation, that gene silencing may occur at the transcriptional level. (c) the cumulative 25-26nt small RNA genes were mainly endemic parasitic Phytophthora genes, including the RXLR effect about 40% genes, 50% genes of Crinkler effect and some elicitor gene, 25-26nt small RNA may play an important role in the new gene silencing and parasitic Phytophthora virulence variation. (d) 25-26nt RNA gene mainly comes from sparse areas and dense areas of genes, and small RNA generation and silence from the repeat region To spread to hundreds of thousands of bases on both sides of the base distance. And this ECHO, endogenous gene RNA located mostly in the accumulation of small or very adjacent area. (E) 21nt RNA from high expression gene, its accumulation is not enough to cause gene silence, and produced a small RNA gene may depend on the expression (f). The re analysis of Phytophthora infestans published little RNA and transcriptome data showed that 25-26nt RNA of Phytophthora infestans, rather than 21nt RNA, and endogenous gene silencing is closely related to the explanation of this phenomenon in Phytophthora is conserved by parasitic.2. the infection of Phytophthora benthamiana (Nicotiana benthamiana) leaves in vitro at different time points (0,3hpi, 6hpi, 12hpi, 24hpi, 48hpi) samples transcriptome sequencing and analysis. The results show that (a) early and late infection stage gene expression differences, a large number of pathogenic Expression of related genes in the early stage of infection in the late expression. Based on this, we identified 231 early infection stage specific expression of genes encoding secreted proteins, which includes a large number of RXLR encoding NPP like effector protein elicitor and various hydrolytic enzymes and protein enzyme inhibitor. It was also found that the gene encoding hydrochloride berberine like protein three gene family of carbonic anhydrase and SCP like extracellular proteins and unknown function gene family in the early infection stage specific expression. We focus on one family system analysis, found that the family members in Phytophthora replication and frequent domain rearrangements significantly. According to the domain arrangement features can be divided into three subfamilies (b). A large number of protein translation, nucleosome assembly and RNA methylation related gene expression was significantly up-regulated in early infection, The expression of three tricarboxylic acid cycle and ATP synthesis related genes in the infection in the late rise. But inconsistent with expectations, vesicle mediated transport, microtubule mediated cell transport, protein degradation, protein phosphorylation and signal transduction related genes significantly down regulated expression of.3. in the infection stage. In addition, we use the transcriptome data of benthamiana gene transcripts were assembled and expression analysis, and through the identification of disease resistance related genes to explore the molecular mechanism of host plant defense of Phytophthora species. The results showed that (a) including a large number of up-regulated gene activity in the oxidative burst and programmed cell necrosis related genes induced by PAMP, and the defense response gene in the infection stage. Salicylic acid, synthesis and signaling pathway related genes in ethylene and jasmonic acid were up-regulated, further analysis showed that these hormone related genes and flg22/e1f18 genes induced by most Expression in the infection stage increases, and two peak expression in 3hpi and 24hpi, which may be an important feature of most genes related to disease resistance. Based on this, we identified 3766 two peak expression genes as candidate genes related to disease resistance in 3hpi and 24hpi, 753 of them in the 3hpi increase more than 10 times. (b) system to identify benthamiana in protein kinases and transcription factors, most of them are 3hpi and 24hpi appeared two peak expression. We further identified to the appearance of the two peak expression of a series of receptor like protein kinase and 4 receptor like protein in 3hpi and 24hpi, provides the important candidate for screening PAMP/ elicitor receptor (d). A large number of proteins, including protein modification and expression of gluconeogenesis, fatty acid oxidation and three tricarboxylic acid cycle, metabolism related genes in the infection stage significantly increased; The related genes such as a large number of biosynthetic pathways, photosynthesis and ion transport were significantly down regulated in protein translation.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S432.4
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