P38天然免疫途徑調(diào)控線蟲對銅綠假單胞菌PA14逃避行為研究
發(fā)布時間：2018-09-19 06:44
【摘要】：土壤中生活的秀麗隱桿線蟲是一種簡單的多細(xì)胞微生物,它們主要以土壤有機細(xì)菌為食物。而細(xì)菌食物又會影響線蟲的多種行為,例如覓食行為、運動行為、趨熱性和趨氣性。土壤中也生存著很多種類的病原細(xì)菌,這些病原細(xì)菌通過線蟲的覓食行為進入到線蟲腸道和角質(zhì)層,進入腸道后病原細(xì)菌可繁殖并殺死線蟲。在長期的生存斗爭中,線蟲學(xué)會感知、并區(qū)分病原細(xì)菌并形成學(xué)習(xí)型逃避行為。P38途徑是秀麗隱桿線蟲最經(jīng)典的一條天然免疫通路。在P38途徑中,tir-1基因同源于哺乳動物的SARM基因,作用于保守信號途徑NSY-1-SEK-1-PMK-1 MAPK的上游。但近期的研究提示：在線蟲抵抗病原菌侵染的過程中,不同的組織中表達(dá)的TIR-1具有不同的作用,分別參與了線蟲對病原菌的天然免疫調(diào)控和逃避行為調(diào)控。雖然在調(diào)控天然免疫的過程中,TIR-1依次激活NSY-1-SEK-1-PMK-1,但tir-1基因在調(diào)控線蟲的逃避行為過程中和下游這些基因之間的作用關(guān)系又如何,該途徑的下游基因是否參與對病原細(xì)菌逃避行為的調(diào)控,還需要進一步闡明�；谠撗芯勘尘�,本論文以秀麗隱桿線蟲為研究對象,利用銅綠假單胞菌(Pseudomonaes aeruginosa,簡稱PA14)菌斑小板逃避實驗,直接檢測P38途徑中不同基因與線蟲病原逃避行為的關(guān)系。并且結(jié)合線蟲雜交、特異神經(jīng)元回復(fù)、熒光定位、表達(dá)譜分析等實驗手段,確定了P38途徑基因在逃避行為中的調(diào)控作用,并且進一步揭示了P38途徑調(diào)控病原細(xì)菌逃避行為的下游通路。本論文主要結(jié)果如下：1.P38途徑中的不同基因?qū)φ{(diào)控PA14的逃避行為具有不同的作用。通過檢測突變株nsy-1(ag3).sek-1(km4)和pmk-1(km25)對PA14的逃避反應(yīng),發(fā)現(xiàn)P38途徑中的NSY-1、SEK-1和PMK-1抑制線蟲的逃避行為；但TIR-1對逃避行為具有正調(diào)控作用。通過構(gòu)建pmk-1 (km25):tir-1(gk264)雙突變株以及pmk-1(km25);Sek-1(km4)雙突變株、并檢測其逃避指數(shù)提示,sek-1、pmk-1位于同一通路；而tir-1與基因sek-1、pmk-1處于平行的通路。并且pmk-1、sek-1和tir-1基因的缺失都不降低線蟲對PA14逃避學(xué)習(xí)能力。此外,直接作用于pm k-1下游的基因Skm-1和atf-7并不參與線蟲逃避行為調(diào)控。2.神經(jīng)元中表達(dá)的PMK-1參與線蟲對PA14的逃避。通過熒光定位研究,證實線蟲的pmk-1基因在腸道和神經(jīng)元均有表達(dá),腸道中表達(dá)的PMK-1調(diào)控線蟲的天然免疫。通過共定位實驗發(fā)現(xiàn)PMK-1表達(dá)部位和TPH-1表達(dá)部位有部分重疊,都在ADF神經(jīng)元中有表達(dá),并且在神經(jīng)元中回復(fù)PMK-1的表達(dá)拯救pmk-1(km2習(xí)突變株對PA14逃避表型,因此神經(jīng)元表達(dá)的PMK-1主要起到調(diào)控線蟲對PA14的逃避行為的作用。此外,對PA14的氣味以及物理致病性傷害的感知是pmnk-1調(diào)控逃避行為所需要的。3.ADF神經(jīng)元中的血清素合成以及RGM神經(jīng)元中的神經(jīng)肽受體NPR-1參與P38途徑對逃避行為的調(diào)控。篩選了依賴和非依賴學(xué)習(xí)逃避行為調(diào)控途徑涉及的相關(guān)基因。經(jīng)過RT-PCR和RNAi干擾實驗初步確定血清素合成相關(guān)基因tph-1和神經(jīng)肽受體npr-1作用于pmnk-1基因下游調(diào)控線蟲的逃避行為。在ADF神經(jīng)元中,pmnk-1基因缺失促使血清素合成增加從而促進線蟲逃避。同時,證實了RMG神經(jīng)元中,pmk-1缺失促進神經(jīng)肽受體NPR-1的表達(dá)激活逃避。并且NPR-]和、TPH-1的作用是平行的,不在同一條分子途徑上。4.Wnt信號途徑和肌動蛋白的相關(guān)基因作用于npr-1下游參與了逃避行為調(diào)控。通過對npr-1基因突變株的RNA-seq表達(dá)譜分析,確定線蟲逃避病原菌PA14過程中,npr-1調(diào)控的與逃避行為相關(guān)的下游基因。通過GO分析和KEGG分析,我們集中篩選了Wnt信號途徑、內(nèi)質(zhì)網(wǎng)應(yīng)激、以及肌動蛋白相關(guān)基因的逃避情況。與野生線蟲N2相比,Wnt途徑的配體own-2正調(diào)控逃避,該基因干擾后逃避指數(shù)降低。卷曲蛋白受體cfz-2正調(diào)控逃避,該基因干擾后線蟲逃避指數(shù)降低。散亂支架蛋白dsh-1正調(diào)逃避,該基因干擾后線蟲逃避指數(shù)降低。受散亂蛋白抑制的糖蛋白激酶gsk-3則是負(fù)調(diào)控逃避的,干擾該基因后逃避指數(shù)增加。此外受糖蛋白激酶抑制的β-catenin合成基因wrm-1正調(diào)控逃避,該基因干擾后逃避指數(shù)降低。肌動蛋白相關(guān)基因msi-1突變導(dǎo)致線蟲的逃避變慢。因此推測金典Wnt信號途和徑肌動蛋白相關(guān)基因作用于npr-1下游參與了逃避行為調(diào)控。論文的創(chuàng)新性：1.首次發(fā)現(xiàn)秀麗隱桿線蟲應(yīng)答病原細(xì)菌侵染時,P38途徑不同基因tir-1、nsy-1、sek-1、pmk-1在調(diào)控逃避行為過程中具有不同的作用。2.證明pmk-1在ADF神經(jīng)元中有表達(dá),神經(jīng)元中表達(dá)的pmnk-1抑制線蟲的逃避行為。此外,對PA14的氣味以及物理致病性傷害的感知是pmnk-1調(diào)控逃避行為必須的。3.證明在線蟲逃避PA14行為中,pmnk-1調(diào)控的下游分子通路包括血清素合成相關(guān)基因tph-1和神經(jīng)肽受體基因npr-1;而Wnt途徑的配體cwn-2、卷曲蛋白受體cfz-2、散亂支架蛋白dsh-1、β-catenin抑制激酶gsk-3、β-catenin合成基因wrm-1、肌動蛋白相關(guān)基因msi-1是潛在的npr-1調(diào)控線蟲對PA14逃避行為的下游基因。
[Abstract]:Caenorhabditis elegans living in the soil is a simple multicellular microorganism that feeds mainly on soil organic bacteria. Bacterial food can affect a variety of behaviors of nematodes, such as foraging behavior, exercise behavior, thermotaxis and aerotaxis. In the long-term survival struggle, nematodes learn to perceive and distinguish pathogenic bacteria and form learning escape behavior. P38 pathway is one of the most classic natural immune pathways of C. elegans. In P38 pathway, tir-1 gene is the same as that of C. elegans. The SARM gene from mammals acts on the upstream of the conserved signaling pathway NSY-1-SEK-1-PMK-1 MAPK. However, recent studies suggest that TIR-1 expressed in different tissues plays different roles in the process of resistance to pathogenic bacteria infection, and is involved in the innate immune regulation and escape behavior of nematodes to pathogenic bacteria. In the process of regulating innate immunity, TIR-1 activates NSY-1-SEK-1-PMK-1 in turn, but what is the relationship between the tir-1 gene and the downstream genes in the process of regulating the escape behavior of nematodes, and whether the downstream genes of this pathway are involved in the regulation of the escape behavior of pathogenic bacteria need further clarification. In this paper, the relationship between different genes in the P38 pathway and the escape behavior of C. elegans was detected by the plaque escape test of Pseudomonas aeruginosa (PA14). The results were confirmed by hybridization, specific neuronal response, fluorescence localization and expression profile analysis. The main results of this study are as follows: 1. Different genes in the P38 pathway have different effects on the escape behavior of PA14. The mutants nsy-1 (ag3). SEK-1 (km4) and pmk-1 (km25) have different effects on the escape behavior of PA14. The escape response of PA14 showed that NSY-1, SEK-1 and PMK-1 in P38 pathway inhibited the escape behavior of nematodes, but TIR-1 had a positive effect on the escape behavior. The deletion of PM k-1, SEK-1 and tir-1 genes did not decrease the ability of nematodes to escape from PA14. In addition, the genes Skm-1 and atf-7 directly acting on the downstream of PM k-1 did not participate in the regulation of nematode escape behavior. 2. PMK-1 expressed in neurons participated in the escape of nematodes from PA14. It was confirmed that the pmk-1 gene was expressed in both intestine and neurons, and the expression of PMK-1 in intestine regulated the innate immunity of the nematode.The co-localization experiment showed that there was a partial overlap between the expression site of PMK-1 and the expression site of TPH-1, both expressed in ADF neurons, and the expression of PMK-1 was reversed in neurons to save pmk-1 (km2 habit mutant strain to PA14). In addition, the perception of PA14 odor and physical pathogenic damage is necessary for pmnk-1 to regulate escape behavior. 3. Serotonin synthesis in ADF neurons and NPR-1 in RGM neurons are involved in P38 pathway. Regulation of evasive behavior. Genes involved in the regulation of dependent and independent learning evasive behavior were screened. RT-PCR and RNAi interference experiments preliminarily confirmed that serotonin synthesis-related genes TPH-1 and neuropeptide receptor NPR-1 acted on the evasive behavior of nematodes downstream of pmnk-1 gene. In ADF neurons, deletion of pmnk-1 promotes blood flow. At the same time, the deletion of pmk-1 promotes the activation and escape of NPR-1 in RMG neurons, and the effects of NPR-] and TPH-1 are parallel, not in the same molecular pathway. 4. Wnt signaling pathway and actin-related genes act on the downstream of NPR-1 and participate in the escape behavior regulation. CONTROL. By analyzing the RNA-seq expression profile of NPR-1 mutant strain, we identified the downstream genes related to escape behavior regulated by NPR-1 in the process of escape from pathogenic bacteria PA14. Through GO analysis and KEGG analysis, we focused on screening the Wnt signaling pathway, endoplasmic reticulum stress, and the escape of actin-related genes. In contrast, the Wnt pathway ligand own-2 positively regulates escape, and the escape index decreases after gene interference. The convolution protein receptor cfz-2 positively regulates escape, and the nematode escape index decreases after gene interference. In addition, wrm-1, the synthesis gene of beta-catenin inhibited by glycoprotein kinase, was regulated to escape, and the escape index decreased after interference. Mutation of actin-related gene Msi-1 resulted in slower escape of nematodes. It was found that tir-1, nsy-1, SEK-1 and pmk-1 genes in P38 pathway played different roles in the regulation of escape behavior. 2. pmk-1 was expressed in ADF neurons and pmnk-1 expressed in neurons inhibited the escape of nematodes. In addition, perception of PA14 odor and physical pathogenic damage is necessary for pmnk-1 to regulate escape behavior. 3. It has been shown that the downstream molecular pathways of pmnk-1 include serotonin synthesis-related genes TPH-1 and neuropeptide receptor gene npr-1, while the Wnt pathway ligands cwn-2, convolution protein receptor cfz-2, are scattered. Scaffold protein dsh-1 and beta-catenin inhibit kinase gsk-3, beta-catenin synthesis gene wrm-1 and actin-related gene Msi-1 are potential downstream genes for NPR-1 to regulate the escape behavior of nematodes to PA14.
【學(xué)位授予單位】：云南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.91
[Abstract]:Caenorhabditis elegans living in the soil is a simple multicellular microorganism that feeds mainly on soil organic bacteria. Bacterial food can affect a variety of behaviors of nematodes, such as foraging behavior, exercise behavior, thermotaxis and aerotaxis. In the long-term survival struggle, nematodes learn to perceive and distinguish pathogenic bacteria and form learning escape behavior. P38 pathway is one of the most classic natural immune pathways of C. elegans. In P38 pathway, tir-1 gene is the same as that of C. elegans. The SARM gene from mammals acts on the upstream of the conserved signaling pathway NSY-1-SEK-1-PMK-1 MAPK. However, recent studies suggest that TIR-1 expressed in different tissues plays different roles in the process of resistance to pathogenic bacteria infection, and is involved in the innate immune regulation and escape behavior of nematodes to pathogenic bacteria. In the process of regulating innate immunity, TIR-1 activates NSY-1-SEK-1-PMK-1 in turn, but what is the relationship between the tir-1 gene and the downstream genes in the process of regulating the escape behavior of nematodes, and whether the downstream genes of this pathway are involved in the regulation of the escape behavior of pathogenic bacteria need further clarification. In this paper, the relationship between different genes in the P38 pathway and the escape behavior of C. elegans was detected by the plaque escape test of Pseudomonas aeruginosa (PA14). The results were confirmed by hybridization, specific neuronal response, fluorescence localization and expression profile analysis. The main results of this study are as follows: 1. Different genes in the P38 pathway have different effects on the escape behavior of PA14. The mutants nsy-1 (ag3). SEK-1 (km4) and pmk-1 (km25) have different effects on the escape behavior of PA14. The escape response of PA14 showed that NSY-1, SEK-1 and PMK-1 in P38 pathway inhibited the escape behavior of nematodes, but TIR-1 had a positive effect on the escape behavior. The deletion of PM k-1, SEK-1 and tir-1 genes did not decrease the ability of nematodes to escape from PA14. In addition, the genes Skm-1 and atf-7 directly acting on the downstream of PM k-1 did not participate in the regulation of nematode escape behavior. 2. PMK-1 expressed in neurons participated in the escape of nematodes from PA14. It was confirmed that the pmk-1 gene was expressed in both intestine and neurons, and the expression of PMK-1 in intestine regulated the innate immunity of the nematode.The co-localization experiment showed that there was a partial overlap between the expression site of PMK-1 and the expression site of TPH-1, both expressed in ADF neurons, and the expression of PMK-1 was reversed in neurons to save pmk-1 (km2 habit mutant strain to PA14). In addition, the perception of PA14 odor and physical pathogenic damage is necessary for pmnk-1 to regulate escape behavior. 3. Serotonin synthesis in ADF neurons and NPR-1 in RGM neurons are involved in P38 pathway. Regulation of evasive behavior. Genes involved in the regulation of dependent and independent learning evasive behavior were screened. RT-PCR and RNAi interference experiments preliminarily confirmed that serotonin synthesis-related genes TPH-1 and neuropeptide receptor NPR-1 acted on the evasive behavior of nematodes downstream of pmnk-1 gene. In ADF neurons, deletion of pmnk-1 promotes blood flow. At the same time, the deletion of pmk-1 promotes the activation and escape of NPR-1 in RMG neurons, and the effects of NPR-] and TPH-1 are parallel, not in the same molecular pathway. 4. Wnt signaling pathway and actin-related genes act on the downstream of NPR-1 and participate in the escape behavior regulation. CONTROL. By analyzing the RNA-seq expression profile of NPR-1 mutant strain, we identified the downstream genes related to escape behavior regulated by NPR-1 in the process of escape from pathogenic bacteria PA14. Through GO analysis and KEGG analysis, we focused on screening the Wnt signaling pathway, endoplasmic reticulum stress, and the escape of actin-related genes. In contrast, the Wnt pathway ligand own-2 positively regulates escape, and the escape index decreases after gene interference. The convolution protein receptor cfz-2 positively regulates escape, and the nematode escape index decreases after gene interference. In addition, wrm-1, the synthesis gene of beta-catenin inhibited by glycoprotein kinase, was regulated to escape, and the escape index decreased after interference. Mutation of actin-related gene Msi-1 resulted in slower escape of nematodes. It was found that tir-1, nsy-1, SEK-1 and pmk-1 genes in P38 pathway played different roles in the regulation of escape behavior. 2. pmk-1 was expressed in ADF neurons and pmnk-1 expressed in neurons inhibited the escape of nematodes. In addition, perception of PA14 odor and physical pathogenic damage is necessary for pmnk-1 to regulate escape behavior. 3. It has been shown that the downstream molecular pathways of pmnk-1 include serotonin synthesis-related genes TPH-1 and neuropeptide receptor gene npr-1, while the Wnt pathway ligands cwn-2, convolution protein receptor cfz-2, are scattered. Scaffold protein dsh-1 and beta-catenin inhibit kinase gsk-3, beta-catenin synthesis gene wrm-1 and actin-related gene Msi-1 are potential downstream genes for NPR-1 to regulate the escape behavior of nematodes to PA14.
【學(xué)位授予單位】：云南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.91
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