【作者】 冯浩；

【导师】 赖仞；

【作者基本信息】 南京农业大学 ， 微生物学， 2009， 硕士

【摘要】 昆虫是地球上种类最多的动物群体,超过100多万种,在生态系统中占据重要地位。一般情况下,昆虫幼虫都是在腐烂的有机物中发育生长,而成虫往往成为导致植物和动物感染疾病的传播载体。因此,昆虫体内逐渐进化出了识别病原微生物和抵御细菌、真菌、寄生虫和病毒感染的能力。除了特殊行为、保护色和机体物理屏障如角质表皮、鳞毛或甲壳外,昆虫还具有识别和清除体内异物的高效免疫系统。免疫系统研究是昆虫研究中最重要的基本问题之一,对于阐明生物的生长发育和起源进化具有重要意义。果蝇(Drosophila melanogaster)由于具有容易饲养、产卵多、生命周期短以及具有粗大的多线染色体而便于进行基因定位等优点,已成为遗传学和发育生物学研究领域中重要的模式生物之一,对现代生命科学的发展作出了突出的贡献。如今果蝇又成为研究昆虫和人类天然免疫的重要模式生物,它主要是通过体液免疫和细胞免疫来抵抗各种病原微生物的侵染。研究表明,果蝇的免疫基因表达主要受两种信号转导途径的调控,即Toll/dif信号转导途径和Imd/Relish信号转导途径。本论文主要以野生型黑腹果蝇为材料,研究了果蝇在受到外界微生物侵染的条件下,其体内Dmloxl2基因的表达变化情况。我们以黑腹果蝇成虫为材料提取其总RNA,然后从中分离出mRNA,建立了果蝇成虫cDNA文库,得到大约为1×106个独立的克隆。这次构建的cDNA文库,为果蝇抗微生物相关基因Dmloxl2的克隆和结构功能研究奠定了基础。我们利用培养至高浓度(600nm下细菌悬液光吸收在50以上)的四种常见微生物(大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌和白色链球菌)人工感染果蝇,以rp49为内参照基因,通过RT-PCR的方法检测果蝇体内Dmloxl2基因的表达变化情况,试验结果显示：黑腹果蝇在受到上述四种微生物侵染12 h后,Dmloxl2基因表达均呈现明显上调趋势。为了进一步验证该实验结果,我们又用实时荧光定量PCR技术检测了Dmloxl2基因在上述四种微生物侵染12 h后的表达变化情况。试验结果显示：果蝇成虫在经过高浓度的E. coli感染时,Dmloxl2的表达上调1.8倍；果蝇成虫在经过高浓度的B. subtilis感染时,Dmloxl2的表达上调2.6倍；果蝇成虫在经过高浓度的S. aureus感染时,Dmloxl2的表达上调2.8倍；果蝇成虫在经过高浓度的C.albicans感染时,Dmloxl2的表达上调2.2倍。上述试验结果说明Dmloxl2基因在受到外界微生物的感染12 h后其表达显著上调,这也进一步验证了前文RT-PCR的实验结果。同时,我们也检测了Dmloxl2基因在上述四种微生物侵染6h后的表达变化情况,其表达也呈现上调趋势。更多还原

【Abstract】 Insect is the largest animal groups on the earth with more than one million species, and play an important role in ecological systems. Frequently, insect larvae develop in decaying organic matter, and insect adults often serve as vectors for microorganisms causing plant and animal diseases. In the process of evolution, insects have formed a highly effective defense system to resist the invasion and parasitization of microbes. Besides the protection of physical barriers such as cuticle, scale and carapace, insects have immune related molecules to recognize and kill invaders. Immune system is one of the most important issues in insect studies and has great significance for clarifying the growth-development and origin.The fruit fly Drosophila melanogaster is an important model organism in research fields of genetics and developmental biology due to the advantages of easy to breed and to get enough embryos in the laboratory, short life cycle, having large polytene chromosomes and so on. It has made great contributions to the development of modern life science.Drosophila has become the important model for the study of the innate immunity of insect and human. Its defense against invading pathogens includes humoral and cellular reactions. The immune gene expression of Drosophila is regulated by two signal transduction pathways:Toll/dif and Imd/relish.In this thesis, under the conditions of microbial infection, the antimicrobial-related gene Dmloxl2 change on expression was studied in wild-type Drosophila melanogaster. cDNA library of Drosophila melanogaster was constructed. 1×106 independent clones were got in the cDNA library. The cDNA library of Drosophila melanogaster, which can improve the structure and function research of antimicrobial-related gene Dmloxl2.We used the high concentration (OD600nm>50) of four microorganisms (Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Candida albicans) infected flies by the method of RT-PCR detection of the expression of Dmloxl2. The results showed that the fruit flies by microbial infection after 12 h, the antimicrobial-related gene Dmloxl2 expression was remarkably up-regulated. To further prove that the experimental results, we had used fluorescence quantitative PCR technique to detect Dmloxl2 gene expression after microbial infection. The results showed that after high concentration of E. coli infection, the expression of Dmloxl2 was increased 1.8; after high concentration of B. subtilis infection, the expression of Dmloxl2 was increased 2.6; after high concentration of S. aureus infection, the expression of Dmloxl2 was increased 2.8; after high concentration of C. albicans infection, the expression of Dmloxl2 was increased 2.2. These results indicated the antimicrobial-related gene Dmloxl2 by microbial infection after 12 h was a significant increase on its expression, which further verified the former RT-PCR results. At the same time, we also detected Dmloxl2 by microbial infection after 6 h, Dmloxl2 expression was also up-regulated.更多还原