P38天然免疫途径调控线虫对铜绿假单胞菌PA14逃避行为研究
发布时间：2018-09-19 06:44
【摘要】：土壤中生活的秀丽隐杆线虫是一种简单的多细胞微生物,它们主要以土壤有机细菌为食物。而细菌食物又会影响线虫的多种行为,例如觅食行为、运动行为、趋热性和趋气性。土壤中也生存着很多种类的病原细菌,这些病原细菌通过线虫的觅食行为进入到线虫肠道和角质层,进入肠道后病原细菌可繁殖并杀死线虫。在长期的生存斗争中,线虫学会感知、并区分病原细菌并形成学习型逃避行为。P38途径是秀丽隐杆线虫最经典的一条天然免疫通路。在P38途径中,tir-1基因同源于哺乳动物的SARM基因,作用于保守信号途径NSY-1-SEK-1-PMK-1 MAPK的上游。但近期的研究提示：在线虫抵抗病原菌侵染的过程中,不同的组织中表达的TIR-1具有不同的作用,分别参与了线虫对病原菌的天然免疫调控和逃避行为调控。虽然在调控天然免疫的过程中,TIR-1依次激活NSY-1-SEK-1-PMK-1,但tir-1基因在调控线虫的逃避行为过程中和下游这些基因之间的作用关系又如何,该途径的下游基因是否参与对病原细菌逃避行为的调控,还需要进一步阐明。基于该研究背景,本论文以秀丽隐杆线虫为研究对象,利用铜绿假单胞菌(Pseudomonaes aeruginosa,简称PA14)菌斑小板逃避实验,直接检测P38途径中不同基因与线虫病原逃避行为的关系。并且结合线虫杂交、特异神经元回复、荧光定位、表达谱分析等实验手段,确定了P38途径基因在逃避行为中的调控作用,并且进一步揭示了P38途径调控病原细菌逃避行为的下游通路。本论文主要结果如下：1.P38途径中的不同基因对调控PA14的逃避行为具有不同的作用。通过检测突变株nsy-1(ag3).sek-1(km4)和pmk-1(km25)对PA14的逃避反应,发现P38途径中的NSY-1、SEK-1和PMK-1抑制线虫的逃避行为；但TIR-1对逃避行为具有正调控作用。通过构建pmk-1 (km25):tir-1(gk264)双突变株以及pmk-1(km25);Sek-1(km4)双突变株、并检测其逃避指数提示,sek-1、pmk-1位于同一通路；而tir-1与基因sek-1、pmk-1处于平行的通路。并且pmk-1、sek-1和tir-1基因的缺失都不降低线虫对PA14逃避学习能力。此外,直接作用于pm k-1下游的基因Skm-1和atf-7并不参与线虫逃避行为调控。2.神经元中表达的PMK-1参与线虫对PA14的逃避。通过荧光定位研究,证实线虫的pmk-1基因在肠道和神经元均有表达,肠道中表达的PMK-1调控线虫的天然免疫。通过共定位实验发现PMK-1表达部位和TPH-1表达部位有部分重叠,都在ADF神经元中有表达,并且在神经元中回复PMK-1的表达拯救pmk-1(km2习突变株对PA14逃避表型,因此神经元表达的PMK-1主要起到调控线虫对PA14的逃避行为的作用。此外,对PA14的气味以及物理致病性伤害的感知是pmnk-1调控逃避行为所需要的。3.ADF神经元中的血清素合成以及RGM神经元中的神经肽受体NPR-1参与P38途径对逃避行为的调控。筛选了依赖和非依赖学习逃避行为调控途径涉及的相关基因。经过RT-PCR和RNAi干扰实验初步确定血清素合成相关基因tph-1和神经肽受体npr-1作用于pmnk-1基因下游调控线虫的逃避行为。在ADF神经元中,pmnk-1基因缺失促使血清素合成增加从而促进线虫逃避。同时,证实了RMG神经元中,pmk-1缺失促进神经肽受体NPR-1的表达激活逃避。并且NPR-]和、TPH-1的作用是平行的,不在同一条分子途径上。4.Wnt信号途径和肌动蛋白的相关基因作用于npr-1下游参与了逃避行为调控。通过对npr-1基因突变株的RNA-seq表达谱分析,确定线虫逃避病原菌PA14过程中,npr-1调控的与逃避行为相关的下游基因。通过GO分析和KEGG分析,我们集中筛选了Wnt信号途径、内质网应激、以及肌动蛋白相关基因的逃避情况。与野生线虫N2相比,Wnt途径的配体own-2正调控逃避,该基因干扰后逃避指数降低。卷曲蛋白受体cfz-2正调控逃避,该基因干扰后线虫逃避指数降低。散乱支架蛋白dsh-1正调逃避,该基因干扰后线虫逃避指数降低。受散乱蛋白抑制的糖蛋白激酶gsk-3则是负调控逃避的,干扰该基因后逃避指数增加。此外受糖蛋白激酶抑制的β-catenin合成基因wrm-1正调控逃避,该基因干扰后逃避指数降低。肌动蛋白相关基因msi-1突变导致线虫的逃避变慢。因此推测金典Wnt信号途和径肌动蛋白相关基因作用于npr-1下游参与了逃避行为调控。论文的创新性：1.首次发现秀丽隐杆线虫应答病原细菌侵染时,P38途径不同基因tir-1、nsy-1、sek-1、pmk-1在调控逃避行为过程中具有不同的作用。2.证明pmk-1在ADF神经元中有表达,神经元中表达的pmnk-1抑制线虫的逃避行为。此外,对PA14的气味以及物理致病性伤害的感知是pmnk-1调控逃避行为必须的。3.证明在线虫逃避PA14行为中,pmnk-1调控的下游分子通路包括血清素合成相关基因tph-1和神经肽受体基因npr-1;而Wnt途径的配体cwn-2、卷曲蛋白受体cfz-2、散乱支架蛋白dsh-1、β-catenin抑制激酶gsk-3、β-catenin合成基因wrm-1、肌动蛋白相关基因msi-1是潜在的npr-1调控线虫对PA14逃避行为的下游基因。
[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.
【学位授予单位】：云南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：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.
【学位授予单位】：云南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q939.91
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