【作者】 刘鑫；

【导师】 覃宏涛； 王冰冰；

【作者基本信息】 湖南大学 ， 生物医学工程（专业学位）， 2021， 硕士

【摘要】 电击作为一种惩罚性刺激,在各类模式生物的研究中有着广泛的应用场景,例如果蝇的应激、学习和记忆等多种行为学研究中,电击都是必不可少的实验条件。然而,目前我们还不是很清楚电击对果蝇基因表达和生理生化方面的影响。本文以转录组测序数据的生物信息学分析为切入点,通过对转录组数据的个性化分析,结合实时荧光定量PCR实验等技术,对电击应激源进行深入探究,并与不同应激源的数据综合分析,探究不同应激源对果蝇影响的异同。本文主要对以下三个方面进行了研究:1.转录组数据分析。将电击处理的果蝇和未电击的果蝇的测序数据进行质量控制、数据清洗,之后进行转录组差异分析。结果表明,果蝇在受到电击的两小时体内的基因表达就发生了显著的改变,共有21个差异上调基因,1829个差异下调基因。富集分析发现,差异上调的基因主要富集在对高氧的反应、对革兰氏阳性和阴性菌的防御反应、抗菌和抗微生物的体液免疫反应以及对细菌的防御反应等通路上,这就说明电击很有可能激活了果蝇体内的免疫相关通路的部分基因。之后的GSEA分析发现体内的Toll、Imd、JAK/STAT通路有上调的趋势,且Toll通路更加明显。在基因表达谱里进一步发现,与果蝇先天免疫息息相关的抗菌肽基因在电击后显著地增强了表达。有意思的是,部分表观遗传相关的基因和转录因子表达出现了显著变化,暗示着电击引起的差异表达可能与染色体表观修饰有关。2.电击对抗菌肽基因表达的调控作用。我们首先验证了实验室的前期发现:电击应激可以诱导抗菌肽基因表达的上调。与对照组相比,电击处理之后雄蝇和处男蝇体内的抗菌肽Metchnikowin(Mtk)和Diptericin(Dpt)表达量均显著上升。随后,我们探究Toll信号通路与电击诱导抗菌肽表达的关系。结果显示,电击对抗菌肽基因表达的诱导作用与Toll通路上游的spz有关。接着,我们发现电击对抗菌肽基因表达的影响可以代间传递。经电击处理的处男蝇与未受电击的处女蝇交配后,其子一代Mtk基因的表达水平比对照组子一代有着显著的提高。但是,抗菌肽的高表达并不能够稳定跨代遗传。到了子二代,电击组和对照组之间就没有差异了。另外经过电击处理的雄蝇在饥饿或无水无食物条件下的存活时间均明显减短,结合测序分析和文献调研,我们推测电击应激会引起果蝇体内的代谢紊乱,且抗菌肽表达是一个高耗能过程,这就导致果蝇对需能逆境的适应性降低。3.不同应激源对果蝇基因表达影响的比较。通过比对不同应激源处理后果蝇的差异基因数据,发现除了抗氧化剂硫化氢,其他应激源条件下果蝇体内的部分抗菌肽基因倾向于上调,且电击与高氧以及衰老条件下的果蝇体内抗菌肽基因的表达多数呈现上调,较为相似。后续对电击、衰老和电离辐射下调的重叠的差异基因进行富集分析,发现这三种条件都影响到了授精、产卵、蛋白质水解的负调控、脂质代谢过程等相关通路。综合测序数据的富集结果,可以看到电击后果蝇体内的众多代谢相关通路都受到了显著影响,这可能表示电击后果蝇的代谢发生了问题。有研究认为果蝇体内抗菌肽基因的高表达是个高耗能的过程;而且在电击独有的差异上调基因中包括了Lime基因,其功能与果蝇体内循环碳水化合物水平的增加、糖原储存减少有关。这暗示着电击应激会引起果蝇体内的代谢紊乱,导致对需能逆境的适应性降低。这一推测获得了本文实验数据的支持,经过电击处理的雄蝇在饥饿或无水无食条件下的存活时间均明显减短。总的来说,果蝇在受到电击后,体内的多数抗菌肽基因表达显著上调,该现象与Toll通路的上游关键基因spz息息相关,且能遗传给下一代。另外,经过电击处理的雄蝇在饥饿或无水无食物条件下的存活时间均明显减短,暗示着电击应激会引起果蝇体内的代谢紊乱,导致对需能逆境的适应性降低。综合对比不同应激源数据,发现电击与衰老和高氧对果蝇的影响有相似之处,部分抗菌肽基因的表达都上调了;而硫化氢对果蝇抗菌肽基因的影响则相反。而电击与衰老和电离辐射都有影响到果蝇的生殖,这也暗示部分应激对果蝇的影响会传递给后代。更多还原

【Abstract】 As a kind of punitive stimulation,electric shock has a wide range of application scenarios in the research of various model organisms.For example,electric shock is an essential experimental condition in the behavioral research of stress,learning and memory of flies.However,the effects of electric shock on gene expression,physiology and biochemistry of Drosophila are still unclear.In this paper,the bioinformatics analysis of transcriptome sequencing data as the breakthrough point,through the personalized analysis of transcriptome data,combined with real-time fluorescence quantitative PCR experiments and other technologies,the shock stressors were deeply explored,and the data of different stressors were comprehensively analyzed to explore the similarities and differences of the effects of different stressors on Drosophila.This paper mainly studies the following three aspects1.Transcriptome data analysis.The sequencing data of Drosophila melanogaster treated with electric shock and non electric shock were used for quality control,data cleaning,and then transcriptome difference analysis.The results showed that there were21 up-regulated genes and 1829 down regulated genes in Drosophila within two hours after electric shock.Enrichment analysis showed that the differentially up-regulated genes were mainly enriched in the response to hyperoxia,the defense response to gram-positive and Gram-negative bacteria,the humoral immune response to antibiotics and microorganisms,and the defense response to bacteria,which indicated that electric shock might activate some genes of immune related pathways in Drosophila.GSEA analysis showed that Toll,Imd and JAK / STAT pathway were up-regulated,and toll pathway was more obvious.In the gene expression profile,it was further found that antimicrobial peptide gene,which is closely related to innate immunity of Drosophila melanogaster,increased significantly after electric shock.Interestingly,the expression of some epigenetic related genes showed significant changes,suggesting that the differential expression caused by electric shock may be related to the epigenetic modification of chromosomes.2.Regulation of antimicrobial peptide gene expression by electric shock.We first verified the previous laboratory findings: shock stress can induce the up regulation of antimicrobial peptide gene expression.Compared with the control group,the expression levels of metchnikowin(MTK)and diptericin(DPT)in male and virgin flies were significantly increased after electric shock treatment.Then,we explored the relationship between Toll signaling pathway and the expression of antimicrobial peptides induced by electric shock.The results showed that the induction of antimicrobial peptide gene expression by electric shock was related to the SPZ upstream of Toll pathway.Then,we found that the effect of electric shock on the expression of antimicrobial peptide gene can be transmitted through generations.The expression level of MTK gene in the offspring of virgin flies treated with electric shock was significantly higher than that of the control group.However,the high expression of antimicrobial peptides can not stabilize the cross generation inheritance.By the second generation,there was no difference between the shock group and the control group.In addition,the survival time of male flies treated with electric shock was significantly shorter under starvation or no food.Combined with sequencing analysis and literature research,we speculated that electric shock stress would cause metabolic disorder in Drosophila melanogaster,and the expression of antimicrobial peptides was a high energy consuming process,which led to the decrease of adaptability of Drosophila melanogaster to energy demand stress.3.Comparison of the effects of different stressors on gene expression of Drosophila melanogaster.By comparing the different gene data of Drosophila treated with different stressors,it was found that except antioxidant hydrogen sulfide,some antimicrobial peptide genes in Drosophila tended to be up-regulated under other stressors,and most antimicrobial peptide genes in Drosophila treated with electric shock,hyperoxia and aging were up-regulated.Subsequent enrichment analysis of the overlapping differential genes down regulated by electric shock,aging and ionizing radiation showed that these three conditions all affected the related pathways such as insemination,oviposition,negative regulation of protein hydrolysis and lipid metabolism.Based on the enrichment results of sequencing data,we can see that many metabolic pathways in Drosophila were significantly affected after electric shock,which may indicate that the metabolism of Drosophila was in trouble after electric shock.Some studies suggest that the high expression of antimicrobial peptide gene in Drosophila is a high energy consuming process;The function of lime gene is related to the increase of circulating carbohydrate level and the decrease of glycogen storage in Drosophila.This suggests that electric shock stress can cause metabolic disorder in Drosophila melanogaster,which leads to the decrease of adaptability to energy demand stress.This hypothesis is supported by the experimental data in this paper.The survival time of male flies treated with electric shock under starvation or drying conditions is significantly reduced.In general,the expression of most antimicrobial peptide genes in Drosophila is significantly up-regulated after electric shock,which is closely related to the upstream key gene SPZ of Toll pathway and can be passed on to the next generation.In addition,the survival time of male flies treated with electric shock was significantly shorter under starvation or no food,which implied that electric shock stress would cause metabolic disorder in Drosophila and reduce their adaptability to energy demand stress.Comparing the data of different stressors,we found that the effects of electric shock,aging and hyperoxia on Drosophila were similar,and the expression of some antimicrobial peptide genes were up-regulated;The effect of hydrogen sulfide on antimicrobial peptide gene of Drosophila was opposite.Electric shock,aging and ionizing radiation can affect the reproduction of Drosophila melanogaster,which also suggests that some of the effects of stress on Drosophila melanogaster will be transmitted to the offspring.更多还原