本文選題：蕓薹屬根腫菌 + 基因組學(xué)��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：蕓薹屬根腫菌(Plasmodiophora brassicae Woron.)是有孔蟲界專性活體寄生植物病原原生動(dòng)物,由其引起的根腫病是十字花科蔬菜和經(jīng)濟(jì)作物上的重要病害,每年造成了重大的經(jīng)濟(jì)損失。國(guó)際上對(duì)根腫菌e3和Pb3菌株進(jìn)行了全基因組序列分析,為深入研究根腫菌生長(zhǎng)、發(fā)育及其致病機(jī)制提供新的契機(jī)�？紤]到根腫菌豐富的致病型,不同致病型或不同地域的菌株之間在基因組上可能會(huì)存在差異,本研究對(duì)分離自我國(guó)湖北省枝江市罹病油菜植株的根腫菌單孢分離菌株ZJ-1菌株(1號(hào)致病型)進(jìn)行了全基因測(cè)序分析,并與e3和Pb3菌株的基因組進(jìn)行了比較,綜合基因組學(xué)和轉(zhuǎn)錄組學(xué),對(duì)根腫菌的分類地位、休眠、生長(zhǎng)和發(fā)育及其致病分子機(jī)理進(jìn)行了系統(tǒng)的分析,并對(duì)生長(zhǎng)、發(fā)育相關(guān)的重要信號(hào)通路和致病因子等進(jìn)行了試驗(yàn)驗(yàn)證,取得的主要研究結(jié)果如下:對(duì)根腫菌ZJ-1菌株進(jìn)行了基因組de novo測(cè)序,獲得的基因組大小為24.1 M,contig N50和scaffold N50分別為73.856 kb和510.27 kb。CEGMA評(píng)價(jià)表明獲得的基因組序列完整性和連續(xù)性好,相近或稍優(yōu)于e3和Pb3的基因組。ZJ-1菌株的基因組大小與e3菌株(24 M)和Pb3菌株(24.2 M)基因組大小相近,但是ZJ-1菌株基因組共預(yù)測(cè)到10,951個(gè)具有蛋白編碼功能的基因,分別比e3和Pb3菌株多出1,221個(gè)和881個(gè)。ZJ-1菌株與e3菌株的基因組共線性高,共有104個(gè)LCBs(局部共線性塊),但也呈現(xiàn)明顯的基因組片段重排(異位、倒置)現(xiàn)象;全基因組點(diǎn)圖比較表明這兩個(gè)基因組也存在明顯的差異。根腫菌ZJ-1菌株基因組重復(fù)序列的總長(zhǎng)度達(dá)為495,571 bp,占全基因組的2.01%,重復(fù)序列比例低、基因密度高,達(dá)到454.40個(gè)基因/Mb。在已測(cè)序的有孔蟲界及SAR超群物種中,根腫菌的基因組最小。將根腫菌與18種已測(cè)序的在進(jìn)化節(jié)點(diǎn)上具代表性的物種進(jìn)行系統(tǒng)進(jìn)化比較,發(fā)現(xiàn)原生生物界的阿米巴蟲和囊泡藻與真菌界、植物界和動(dòng)物界聚為一支,而包括根腫菌在內(nèi)的有孔蟲界物種成為真核生物進(jìn)化樹中獨(dú)立的一支,表明以根腫菌為代表的有孔蟲界物種在真核生物進(jìn)化史中的獨(dú)特進(jìn)化地位。從ZJ-1菌株看,根腫菌物種獨(dú)有的基因家族基因共4,197個(gè);117基因家族,共計(jì)611個(gè)基因顯著擴(kuò)張。對(duì)這些基因的GO功能富集分析表明其功能主要富集于GPCR信號(hào)轉(zhuǎn)導(dǎo)途徑、幾丁質(zhì)生物合成過(guò)程和DNA整合等生物學(xué)過(guò)程等。推定這些過(guò)程對(duì)根腫菌發(fā)育至關(guān)重要。通過(guò)GPCR抑制劑處理,抑制根腫菌GPCR相關(guān)通路,可以延緩根腫菌的發(fā)育進(jìn)程,顯著緩解根系腫大癥狀,進(jìn)一步證實(shí)GPCR信號(hào)轉(zhuǎn)導(dǎo)相關(guān)通路在根腫菌生長(zhǎng)發(fā)育和致病過(guò)程中具有重要的作用。從多組學(xué)角度對(duì)處于休眠孢子階段、休眠孢子萌發(fā)階段、次生原質(zhì)團(tuán)發(fā)育階段三個(gè)不同生長(zhǎng)發(fā)育狀態(tài)的根腫菌進(jìn)行了RNA-Seq測(cè)序分析,對(duì)這三個(gè)狀態(tài)下的基因調(diào)控分子機(jī)理進(jìn)行深入解析。發(fā)現(xiàn)在甾體降解代謝、類黃酮生物合成、α-亞麻酸代謝、甘油磷脂代謝和醚脂類代謝途徑等在根腫菌休眠孢子中比較活躍,推定它們與根腫菌維持休眠狀態(tài)緊密相關(guān);在休眠孢子萌發(fā)階段,病菌可能需要激活更多碳水化合物代謝通路。次生原質(zhì)團(tuán)發(fā)育階段的基因表達(dá)調(diào)控模式不同于另外兩個(gè)階段,該階段有458個(gè)基因顯著上調(diào)表達(dá),它們參與細(xì)胞分裂、生長(zhǎng)、DNA復(fù)制和蛋白翻譯等過(guò)程相關(guān)。在休眠孢子萌發(fā)階段和次生原質(zhì)團(tuán)發(fā)育階段中含錨蛋白重復(fù)結(jié)構(gòu)域、含P環(huán)三磷酸核苷水解酶和絲氨酸/蘇氨酸蛋白激酶等等基因的表達(dá)量均很高。休眠孢子對(duì)于根腫菌的存活和傳播十分重要,休眠孢子中脂滴的成分與孢子的存活是否相關(guān)并不清楚。從多組學(xué)角度分析了在根腫菌中的脂滴及其相關(guān)的代謝途徑。利用蛋白質(zhì)組學(xué)分析手段從根腫菌休眠孢子脂滴中鑒定出295個(gè)脂滴結(jié)合蛋白,從基因組中鑒定出167個(gè)參與脂代謝相關(guān)通路的基因,脂肪酸合成途徑相關(guān)的基因在次生原質(zhì)團(tuán)發(fā)育階段表達(dá)水平高;在休眠孢子階段,花生四烯酸代謝途徑、醚脂類代謝、酮體合成和降解途徑的基因具有較高表達(dá)量。根腫菌富含甘油三脂,ZJ-1菌株基因組中包含有完整的甘油三酯合成、儲(chǔ)存和降解通路�；虮磉_(dá)動(dòng)態(tài)的聚類分析表明處于休眠孢子萌發(fā)階段的根腫菌通過(guò)甘油三酯和脂肪酸降解過(guò)程,利用脂滴中的貯存的能量;休眠孢子中大部分(76.6%)的脂肪酸屬于飽和脂肪酸(C16:0和C18:0),不飽和脂肪酸中花生四烯酸C20:4含量最高,占整個(gè)不飽和脂肪酸含量50%左右。根腫菌是植物細(xì)胞內(nèi)的活體寄生菌,這種寄生方法在植物真核病原物中非常獨(dú)特,一方面需要維系寄主細(xì)胞的活力,同時(shí)也需要成功抑制寄主的防御反應(yīng)。我們對(duì)這種專性活體寄生生活方式從比較基因組學(xué)、轉(zhuǎn)錄組學(xué)和分泌組學(xué)角度進(jìn)行了解析。發(fā)現(xiàn)許多參與初級(jí)代謝過(guò)程的基因,在根腫菌進(jìn)化過(guò)程中丟失;根腫菌基因組中CAZymes相關(guān)基因和次級(jí)代謝基因減少,體現(xiàn)其極端的活體寄生策略;編碼ABC轉(zhuǎn)運(yùn)體的基因在基因組中顯著擴(kuò)張且相關(guān)基因在次生原質(zhì)團(tuán)發(fā)育階段具有較高表達(dá)水平。ZJ-1菌株中至少有4個(gè)有參與調(diào)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,推定它們編碼的蛋白參與了寄主植物激素的調(diào)節(jié)過(guò)程。從ZJ-1菌株基因組中預(yù)測(cè)到739個(gè)編碼分泌蛋白的基因,其中111個(gè)基因在休眠孢子萌發(fā)階段或次生原質(zhì)團(tuán)生長(zhǎng)階段具有較高的表達(dá)水平。最后篩選出20個(gè)候選效應(yīng)子蛋白,其中8個(gè)為ZJ-1菌株特有。選取其中6個(gè)候選效應(yīng)子蛋白進(jìn)行進(jìn)一步驗(yàn)證,發(fā)現(xiàn)它們均可分泌到胞外;在本氏煙草上測(cè)定結(jié)果表明效應(yīng)子蛋白EC4(Plas B_07397)能誘導(dǎo)煙草葉片產(chǎn)生嚴(yán)重壞死癥狀,且其質(zhì)外體定位對(duì)于其引起煙草葉片的壞死反應(yīng)至關(guān)重要;效應(yīng)子蛋白EC1(PlasB_06695)能夠抑制由促凋亡蛋白Bax誘導(dǎo)的煙草葉片壞死反應(yīng)。這些特性均揭示了根腫菌專性活體寄生的獨(dú)特方式。根腫菌次級(jí)游動(dòng)孢子進(jìn)入寄主皮層細(xì)胞后,發(fā)育成次生原質(zhì)團(tuán)。原質(zhì)團(tuán)的細(xì)胞核不斷分裂,其體積也不斷增大,最終原質(zhì)團(tuán)割裂,形成成熟的休眠孢子充滿寄主細(xì)胞。次生原質(zhì)團(tuán)發(fā)育階段差異表達(dá)的基因,其KEGG途徑功能主要富集于“翻譯”、“細(xì)胞生長(zhǎng)與死亡”、“細(xì)胞通訊”和“癌癥”四個(gè)方面。以此為線索,從ZJ-1菌株全基因組中鑒定出171個(gè)能夠注釋到與癌癥相關(guān)途徑的基因,其中原癌基因Pb-Raf1、Pb-Raf2和Pb-MYB編碼的蛋白分別與人類原癌基因Raf、MYB同源,且它們?cè)诖紊|(zhì)團(tuán)發(fā)育階段被顯著激活;鑒定出三個(gè)與癌癥信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的通路(Ras/Raf/MEK/ERK信號(hào)通路、PI3K/Akt信號(hào)通路、mTOR信號(hào)通路),在多核次生原質(zhì)團(tuán)發(fā)育階段,信號(hào)轉(zhuǎn)導(dǎo)通路中的12個(gè)核心基因也具有顯著上調(diào)表達(dá)趨勢(shì)。利用PI3K抑制劑抑制根腫菌的PI3K/Akt信號(hào)通路后發(fā)現(xiàn)次生原質(zhì)團(tuán)發(fā)育進(jìn)程顯著受阻,緩解了油菜的根腫癥狀。進(jìn)一步表明癌癥相關(guān)PI3K信號(hào)轉(zhuǎn)導(dǎo)通路在根腫菌次生原質(zhì)團(tuán)發(fā)育階段具有重要的作用。綜上所述,本研究對(duì)根腫菌1號(hào)致病型ZJ-1菌株進(jìn)行了全基因組de novo測(cè)序分析,解析了根腫菌的休眠、生長(zhǎng)、發(fā)育和致病等生命活動(dòng),研究結(jié)果對(duì)建立根腫病的綠色防控技術(shù)提供了新的研究線索。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S432.4

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