多组学研究揭示根肿菌生长发育与致病的分子机理

发布时间：2018-04-23 22:12

本文选题：芸薹属根肿菌 + 基因组学　；参考：《华中农业大学》2017年博士论文

【摘要】：芸薹属根肿菌(Plasmodiophora brassicae Woron.)是有孔虫界专性活体寄生植物病原原生动物,由其引起的根肿病是十字花科蔬菜和经济作物上的重要病害,每年造成了重大的经济损失。国际上对根肿菌e3和Pb3菌株进行了全基因组序列分析,为深入研究根肿菌生长、发育及其致病机制提供新的契机。考虑到根肿菌丰富的致病型,不同致病型或不同地域的菌株之间在基因组上可能会存在差异,本研究对分离自我国湖北省枝江市罹病油菜植株的根肿菌单孢分离菌株ZJ-1菌株(1号致病型)进行了全基因测序分析,并与e3和Pb3菌株的基因组进行了比较,综合基因组学和转录组学,对根肿菌的分类地位、休眠、生长和发育及其致病分子机理进行了系统的分析,并对生长、发育相关的重要信号通路和致病因子等进行了试验验证,取得的主要研究结果如下:对根肿菌ZJ-1菌株进行了基因组de novo测序,获得的基因组大小为24.1 M,contig N50和scaffold N50分别为73.856 kb和510.27 kb。CEGMA评价表明获得的基因组序列完整性和连续性好,相近或稍优于e3和Pb3的基因组。ZJ-1菌株的基因组大小与e3菌株(24 M)和Pb3菌株(24.2 M)基因组大小相近,但是ZJ-1菌株基因组共预测到10,951个具有蛋白编码功能的基因,分别比e3和Pb3菌株多出1,221个和881个。ZJ-1菌株与e3菌株的基因组共线性高,共有104个LCBs(局部共线性块),但也呈现明显的基因组片段重排(异位、倒置)现象;全基因组点图比较表明这两个基因组也存在明显的差异。根肿菌ZJ-1菌株基因组重复序列的总长度达为495,571 bp,占全基因组的2.01%,重复序列比例低、基因密度高,达到454.40个基因/Mb。在已测序的有孔虫界及SAR超群物种中,根肿菌的基因组最小。将根肿菌与18种已测序的在进化节点上具代表性的物种进行系统进化比较,发现原生生物界的阿米巴虫和囊泡藻与真菌界、植物界和动物界聚为一支,而包括根肿菌在内的有孔虫界物种成为真核生物进化树中独立的一支,表明以根肿菌为代表的有孔虫界物种在真核生物进化史中的独特进化地位。从ZJ-1菌株看,根肿菌物种独有的基因家族基因共4,197个;117基因家族,共计611个基因显著扩张。对这些基因的GO功能富集分析表明其功能主要富集于GPCR信号转导途径、几丁质生物合成过程和DNA整合等生物学过程等。推定这些过程对根肿菌发育至关重要。通过GPCR抑制剂处理,抑制根肿菌GPCR相关通路,可以延缓根肿菌的发育进程,显著缓解根系肿大症状,进一步证实GPCR信号转导相关通路在根肿菌生长发育和致病过程中具有重要的作用。从多组学角度对处于休眠孢子阶段、休眠孢子萌发阶段、次生原质团发育阶段三个不同生长发育状态的根肿菌进行了RNA-Seq测序分析,对这三个状态下的基因调控分子机理进行深入解析。发现在甾体降解代谢、类黄酮生物合成、α-亚麻酸代谢、甘油磷脂代谢和醚脂类代谢途径等在根肿菌休眠孢子中比较活跃,推定它们与根肿菌维持休眠状态紧密相关;在休眠孢子萌发阶段,病菌可能需要激活更多碳水化合物代谢通路。次生原质团发育阶段的基因表达调控模式不同于另外两个阶段,该阶段有458个基因显著上调表达,它们参与细胞分裂、生长、DNA复制和蛋白翻译等过程相关。在休眠孢子萌发阶段和次生原质团发育阶段中含锚蛋白重复结构域、含P环三磷酸核苷水解酶和丝氨酸/苏氨酸蛋白激酶等等基因的表达量均很高。休眠孢子对于根肿菌的存活和传播十分重要,休眠孢子中脂滴的成分与孢子的存活是否相关并不清楚。从多组学角度分析了在根肿菌中的脂滴及其相关的代谢途径。利用蛋白质组学分析手段从根肿菌休眠孢子脂滴中鉴定出295个脂滴结合蛋白,从基因组中鉴定出167个参与脂代谢相关通路的基因,脂肪酸合成途径相关的基因在次生原质团发育阶段表达水平高;在休眠孢子阶段,花生四烯酸代谢途径、醚脂类代谢、酮体合成和降解途径的基因具有较高表达量。根肿菌富含甘油三脂,ZJ-1菌株基因组中包含有完整的甘油三酯合成、储存和降解通路。基因表达动态的聚类分析表明处于休眠孢子萌发阶段的根肿菌通过甘油三酯和脂肪酸降解过程,利用脂滴中的贮存的能量;休眠孢子中大部分(76.6%)的脂肪酸属于饱和脂肪酸(C16:0和C18:0),不饱和脂肪酸中花生四烯酸C20:4含量最高,占整个不饱和脂肪酸含量50%左右。根肿菌是植物细胞内的活体寄生菌,这种寄生方法在植物真核病原物中非常独特,一方面需要维系寄主细胞的活力,同时也需要成功抑制寄主的防御反应。我们对这种专性活体寄生生活方式从比较基因组学、转录组学和分泌组学角度进行了解析。发现许多参与初级代谢过程的基因,在根肿菌进化过程中丢失;根肿菌基因组中CAZymes相关基因和次级代谢基因减少,体现其极端的活体寄生策略;编码ABC转运体的基因在基因组中显著扩张且相关基因在次生原质团发育阶段具有较高表达水平。ZJ-1菌株中至少有4个有参与调控寄主植物激素水平的基因,其中PBHK1和PBHK2在ZJ-1和e3菌株中均存在,推定它们编码的蛋白参与了寄主植物激素的调节过程。从ZJ-1菌株基因组中预测到739个编码分泌蛋白的基因,其中111个基因在休眠孢子萌发阶段或次生原质团生长阶段具有较高的表达水平。最后筛选出20个候选效应子蛋白,其中8个为ZJ-1菌株特有。选取其中6个候选效应子蛋白进行进一步验证,发现它们均可分泌到胞外;在本氏烟草上测定结果表明效应子蛋白EC4(Plas B_07397)能诱导烟草叶片产生严重坏死症状,且其质外体定位对于其引起烟草叶片的坏死反应至关重要;效应子蛋白EC1(PlasB_06695)能够抑制由促凋亡蛋白Bax诱导的烟草叶片坏死反应。这些特性均揭示了根肿菌专性活体寄生的独特方式。根肿菌次级游动孢子进入寄主皮层细胞后,发育成次生原质团。原质团的细胞核不断分裂,其体积也不断增大,最终原质团割裂,形成成熟的休眠孢子充满寄主细胞。次生原质团发育阶段差异表达的基因,其KEGG途径功能主要富集于“翻译”、“细胞生长与死亡”、“细胞通讯”和“癌症”四个方面。以此为线索,从ZJ-1菌株全基因组中鉴定出171个能够注释到与癌症相关途径的基因,其中原癌基因Pb-Raf1、Pb-Raf2和Pb-MYB编码的蛋白分别与人类原癌基因Raf、MYB同源,且它们在次生原质团发育阶段被显著激活;鉴定出三个与癌症信号转导相关的通路(Ras/Raf/MEK/ERK信号通路、PI3K/Akt信号通路、mTOR信号通路),在多核次生原质团发育阶段,信号转导通路中的12个核心基因也具有显著上调表达趋势。利用PI3K抑制剂抑制根肿菌的PI3K/Akt信号通路后发现次生原质团发育进程显著受阻,缓解了油菜的根肿症状。进一步表明癌症相关PI3K信号转导通路在根肿菌次生原质团发育阶段具有重要的作用。综上所述,本研究对根肿菌1号致病型ZJ-1菌株进行了全基因组de novo测序分析,解析了根肿菌的休眠、生长、发育和致病等生命活动,研究结果对建立根肿病的绿色防控技术提供了新的研究线索。
[Abstract]:Plasmodiophora brassicae Woron. is the pathogenic protozoa of the special living parasitic plant of the foraminifera. The root swelling caused by it is an important disease on cruciferous vegetables and economic crops. It has caused great economic loss every year. The whole genome sequence analysis of E3 and Pb3 strains of root swollen bacteria was carried out in the world. It provides a new opportunity to study the growth, development and pathogenesis of rhizomaria. Taking into account the rich pathogenicity of rhizomaria, there may be differences in genomes between strains of different pathogenicity or different regions. In this study, the single spore isolate ZJ-1 strain ZJ-1 (No. 1) isolated from the infected rapeseed plants in Zhijiang, China (1) The whole gene sequencing analysis was carried out and compared with the genomes of E3 and Pb3 strains. Comprehensive genomics and transcripology were used to systematically analyze the classification status, dormancy, growth and development and the molecular mechanism of the bacteria, and to test the growth, the important signaling pathways and the pathogenic factors related to the development of the bacteria. The main results of the study were as follows: genomic de novo sequencing was carried out for ZJ-1 strains of root swell bacteria, the size of genome was 24.1 M, contig N50 and scaffold N50 were 73.856 KB and 510.27 kb.CEGMA respectively, which showed that the integrity and continuity of the genome sequence were good, similar to or slightly better than E3 and Pb3. The genome size of the strain was similar to the size of the E3 strain (24 M) and the Pb3 strain (24.2 M). But the genome of the ZJ-1 strain was predicted to 10951 genes with protein coding function, and 1221 and 881.ZJ-1 strains were higher than the E3 and Pb3 strains, respectively, and there were 104 LCBs (local collinear blocks). The total genomic point map showed obvious differences between the two genomes. The total genome length of the two genomes was 495571 BP, 2.01% of the whole genome, the low repeat sequence and the high gene density, and 454.40 gene /Mb. were sequenced. The genomes of the foraminifera and SAR species are the smallest. Compare the phylogenetic relationship with 18 sequenced representative species on the evolutionary nodes, it is found that the amoeba and vesicles of the primary biological boundary are associated with the fungal, plant and animal boundaries, and the foraminifera, including the root swell bacteria. The species became an independent branch of the eukaryote evolution tree, indicating the unique evolutionary status of the foraminifera species represented by the swell bacteria in the evolutionary history of eukaryotes. From the ZJ-1 strain, there are 4197 gene family genes unique to the swell species; the 117 gene family, with a total of 611 bases, has expanded significantly. The GO function enrichment of these genes is enriched. The analysis shows that its function is mainly enriched in GPCR signal transduction pathway, biosynthesis process of chitin biosynthesis and DNA integration and other biological processes. It is concluded that these processes are important for the development of root swell bacteria. Through the treatment of GPCR inhibitors, the inhibition of the GPCR related pathway of root swelling bacteria can delay the development of root swelling bacteria and significantly alleviate the symptoms of root swelling. The GPCR signal transduction pathway plays an important role in the growth and pathogenesis of root swell bacteria. The RNA-Seq sequencing analysis of three different growth and development stages in the dormant spore stage, the dormant spore germination stage and the secondary Plasmodium development stage has been carried out from the multi group perspective, and the bases under these three states are analyzed. It was found that in steroid Degradation Metabolism, flavonoid biosynthesis, alpha linolenic metabolism, glycerol phospholipid metabolism and ether lipid metabolism pathway are more active in the dormant spore of root swollen bacteria, presumed that they are closely related to the dormancy state of root swollen bacteria, and the bacteria may be stimulated at the stage of dormant spore germination. The regulation patterns of gene expression in the developmental stage of secondary Plasmodium are different from the other two stages, and 458 genes are significantly up-regulated at this stage. They are involved in the process of cell division, growth, DNA replication and protein translation. The expression of the P ring three phosphoric acid nucleoside hydrolase and serine / threonine protein kinase and other genes are high. The dormant spores are very important for the survival and propagation of the bacteria. Whether the lipid droplets in the dormant spores are related to the survival of the spores are not clear. 295 lipoprotein binding proteins were identified by proteomic analysis, and 167 genes involved in lipid metabolism related pathways were identified from the genome. The genes related to the fatty acid synthesis pathway expressed high levels in the secondary Plasmodium development stage; in the dormant spores, the genes related to the fatty acid synthesis pathway were high in the developmental stage of secondary Plasmodium. In the substage, the genes of peanut four enaconic acid metabolism, ether lipid metabolism, ketone body synthesis and degradation pathway have high expression. The genomes of ZJ-1 are rich in glycerol three fat, and the genome contains a complete triglyceride synthesis, storage and degradation pathway. The bacteria use the triglyceride and fatty acid degradation process and use the energy stored in the lipid droplets; most of the (76.6%) fatty acids in the dormant spores belong to saturated fatty acids (C16:0 and C18:0), and the highest content of arachidonic acid C20:4 in unsaturated fatty acids, accounting for about 50% of the total unsaturated fatty acid, is the living parasitic of plant cells. This parasitic method is very unique in plant eukaryotic organisms. On the one hand, it is necessary to maintain the vitality of host cells and also to inhibit the host's defensive response. We have analyzed this specific living lifestyle from the perspective of comparative genomics, transcripology and secretory histology. Many of them have been found to be involved in the primary generation. The gene of the metabolic process is lost during the evolution of the root of the root swollen bacteria; the CAZymes related genes and secondary metabolites in the genomes of the genomes decrease, reflecting their extreme living parasitic strategies; the genes encoding the ABC transporters in the genome are significantly expanded in the genome and the related genes are in the higher expression level of.ZJ-1 strains in the secondary Plasmodium development stage. There are 4 few genes involved in regulating host plant hormone levels, in which PBHK1 and PBHK2 exist in both ZJ-1 and E3 strains. It is presumed that their encoded proteins are involved in the regulation of host plant hormones, and 739 genes encoding secretory proteins are predicted from the genome of ZJ-1 strain, of which 111 are at the stage of dormant spore germination or secondary. 20 candidate effect subproteins were selected, of which 8 were specific to ZJ-1, and 6 of them were selected for further validation, and they were found to be secreted out of the cell. The results of the determination of the effector protein EC4 (Plas B_07397) could induce smoke in the tobacco. Grass leaves produce severe necrotic symptoms, and the location of the ectobody is crucial to the necrosis of tobacco leaves. The effector protein EC1 (PlasB_06695) can inhibit the leaf necrosis induced by the apoptotic protein Bax. These characteristics reveal the unique way of the parasitic parasitism of the bacteria. After entering the cells of the host cortex, the cells develop into secondary Plasmodium. The nuclei of the Plasmodium continue to split and their volume is increasing, and eventually the Plasmodium is split and the mature dormant spores are filled with host cells. The differentially expressed genes in the developmental stages of the secondary Plasmodium are mainly enriched in the "translation", "the cell growth and the growth of the KEGG pathway." Death "," cell communication "and" cancer "four aspects. From the whole genome of the ZJ-1 strain, 171 genes were identified to be annotated to cancer related pathways, in which the proto oncogene Pb-Raf1, Pb-Raf2 and Pb-MYB encoded proteins were homologous to the human proto oncogene Raf, MYB, and they were in the development stage of the secondary Plasmodium. Three pathways associated with cancer signal transduction (Ras/Raf/MEK/ERK signaling pathway, PI3K/Akt signaling pathway, mTOR signaling pathway) were identified, and the 12 core genes in the signal transduction pathway were also significantly up-regulated in the developmental stages of the multicore secondary Plasmodium. PI3K inhibitors were used to inhibit the PI3K/Akt signal of the bacteria. It was found that the development process of secondary Plasmodium was significantly hindered and alleviated the root swelling symptoms of rapeseed. It was further indicated that the cancer related PI3K signal transduction pathway played an important role in the development stage of the secondary plasmodium of root swell. To sum up, the whole genome de novo sequencing of the 1 pathogenic ZJ-1 strains of root swell bacteria was analyzed. The dormancy, growth, development and pathogenicity of swollen fungi were analyzed, and the results provided new clues for the establishment of green prevention and control technology for root knot disease.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S432.4

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