miR-981在果蝇Imd免疫响应中的调控作用研究
发布时间：2018-07-18 08:56
【摘要】：黑腹果蝇作为重要的生物学研究模式动物之一,在研究基因表达调控、神经科学、人类疾病及遗传机制等多个研究领域发挥出了重要作用。目前,有关果蝇的先天性免疫方面的研究已经取得了诸多的成就,主要包括体液免疫和细胞免疫,其中Toll和Imd是果蝇最重要的体液免疫方式,并在近些年的研究过程中鉴定出了很多参与免疫响应的基因。MicroRNA (miRNA)是非编码小RNA家族的一员,长度约为21-24nt的单链小分子RNA。miRNA可以通过靶向一个或多个靶基因,并且抑制靶基因的翻译水平来调节基因的表达。而在果蝇体内,miRNA是否会通过与免疫基因相互作用来调节免疫响应,一直是我们感兴趣的问题。目前,只有极少的miRNA被发现参与调控果蝇的免疫应答。本论文主要研究的是miR-981对于果蝇Imd免疫信号通路的调控机制,所采取的实验研究和所得结果如下:1.通过革兰氏阴性菌刺激野生型果蝇,然后利用qRT-PCR技术检测3h、6h、12h、24h、48h和72h果蝇体内的miR-981和Imd信号通路的代表性抗菌肽Diptericin的表达量。结果发现,在抗菌肽Diptericin的表达量先由上升随后逐渐降低的整个应答过程中,miR-981的表达量在6h和12h表现出显著的升高,这些结果暗示果蝇Imd信号的激活引起了 miR-981表达量的变化。2.利用Tub-Ga180ts/+;Tub-Ga14系统果蝇与UAS-miR-981果蝇杂交,得到miR-981高表达的果蝇株。通过免疫损伤实验发现,在miR-981高表达果蝇体内,Diptericin的表达量表现出显著的下降。然而Attain、Cecropin和Defensin等其他抗菌肽的表达量却没有明显的差异,这些结果暗示miR-981可能直接参与调控抗菌肽Diptericin的表达来调节果蝇Imd信号响应的免疫应答。3.为了进一步验证miR-981和Diptericin之间的作用关系,我们先通过TargetScan和miRanda两种生物信息学预测软件预测了Dipteri in和miR-981之间的靶位点信息。预测结果均表明,miR-981和Diptericin存在着直接的靶作用关系。4.为了证实miR-981和DDiptericin的作用关系,我们在体外采用双荧光素酶报告系统在果蝇S2细胞内进行进一步研究,结果表明miR-981确实能够抑制Diptericin的表达,而当我们将二者之间的作用靶位点进行突变后,miR-981对Diptericin的抑制作用就不存在了,这表明miR-981直接抑制了Dipterici 的表达。5.随后,我们在体内将miR-981高表达果蝇体内的miR-981表达量降至正常水平。在miR-981的表达量降低后,再次检测的结果显示,Diptericin的表达量与对照组相比没有显著的差别。综上所述,我们采用生物学的实验研究手段以及生物信息学的分析方法,研究证明了黑腹果蝇miR-981可以通过直接靶向Diptericin的3'UTR序列进而调控果蝇的Imd免疫信号通路。本论文的研究为探讨miRNA参与果蝇免疫调控提供了参考。
[Abstract]:Drosophila melanogaster, as one of the important biological research model animals, plays an important role in many research fields, such as gene expression regulation, neuroscience, human disease and genetic mechanism. At present, many achievements have been made in the study of congenital immunity of Drosophila melanogaster, mainly including humoral immunity and cellular immunity, of which Toll and IMD are the most important humoral immunity of Drosophila melanogaster. In recent years, we have identified many genes involved in immune response. MicroRNA (miRNA) is a member of non-coding small RNA family, and the length of single stranded RNA.miRNA can be targeted at one or more target genes. The expression of target gene was regulated by inhibiting the translation level of target gene. The question of whether miRNA interacts with immune genes to regulate immune response in Drosophila melanogaster has always been a question of interest to us. At present, very few miRNAs have been found to be involved in regulating the immune response of Drosophila melanogaster. In this thesis, we mainly studied the regulation mechanism of miR-981 on IMD immune signaling pathway in Drosophila melanogaster. The experimental results are as follows: 1. The wild-type Drosophila melanogaster was stimulated by Gram-negative bacteria. The expression of diptericin, a representative antimicrobial peptide of miR-981 and IMD signaling pathway in Drosophila melanogaster for 48 h and 72 h, was detected by qRT-PCR. The results showed that the expression of diptericin increased at 6h and 12h during the whole response. These results suggested that the activation of IMD signal in Drosophila caused the change of miR-981 expression. The highly expressed Drosophila strain miR-981 was obtained by crossing between Drosophila melanogaster and UAS-miR-981 by Tub-Ga180ts/ Tub-Ga14 system. The expression of Diptericin in the highly expressed Drosophila melanogaster miR-981 showed a significant decrease. However, there was no significant difference in the expression of other antimicrobial peptides such as Cecropin and Defensin. These results suggest that miR-981 may be directly involved in regulating the expression of the antimicrobial peptide Diptericin to regulate the immune response of Drosophila IMD signal. In order to further verify the interaction between miR-981 and Diptericin, the target information between Dipteri in and miR-981 was predicted by TargetScan and miRanda bioinformatics prediction software. The predicted results showed that there was a direct target action relationship between diptericin and miR-981. In order to confirm the relationship between miR-981 and DDiptericin, we used double luciferase report system in vitro to further study the expression of Diptericin in S2 cells of Drosophila melanogaster. The results showed that miR-981 could inhibit the expression of Diptericin. The inhibitory effect of miR-981 on Diptericin did not exist when we mutated the target sites between them, which indicated that miR-981 directly inhibited the expression of Dipterici. Subsequently, we reduced the expression of miR-981 to normal level in drosophila melanogaster. After the expression of miR-981 was decreased, the results of re-detection showed that there was no significant difference in the expression of diptericin between the control group and the control group. To sum up, we used biological experimental methods and bioinformatics analysis methods to prove that miR-981 can regulate the IMD immune signal pathway of Drosophila melanogaster by directly targeting Diptericin's 3G UTR sequence and then regulating the IMD immune signal pathway of Drosophila melanogaster (Drosophila melanogaster). This study provides a reference for the study of miRNA involved in the immune regulation of Drosophila melanogaster.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392;R-332
本文编号：2131403
[Abstract]:Drosophila melanogaster, as one of the important biological research model animals, plays an important role in many research fields, such as gene expression regulation, neuroscience, human disease and genetic mechanism. At present, many achievements have been made in the study of congenital immunity of Drosophila melanogaster, mainly including humoral immunity and cellular immunity, of which Toll and IMD are the most important humoral immunity of Drosophila melanogaster. In recent years, we have identified many genes involved in immune response. MicroRNA (miRNA) is a member of non-coding small RNA family, and the length of single stranded RNA.miRNA can be targeted at one or more target genes. The expression of target gene was regulated by inhibiting the translation level of target gene. The question of whether miRNA interacts with immune genes to regulate immune response in Drosophila melanogaster has always been a question of interest to us. At present, very few miRNAs have been found to be involved in regulating the immune response of Drosophila melanogaster. In this thesis, we mainly studied the regulation mechanism of miR-981 on IMD immune signaling pathway in Drosophila melanogaster. The experimental results are as follows: 1. The wild-type Drosophila melanogaster was stimulated by Gram-negative bacteria. The expression of diptericin, a representative antimicrobial peptide of miR-981 and IMD signaling pathway in Drosophila melanogaster for 48 h and 72 h, was detected by qRT-PCR. The results showed that the expression of diptericin increased at 6h and 12h during the whole response. These results suggested that the activation of IMD signal in Drosophila caused the change of miR-981 expression. The highly expressed Drosophila strain miR-981 was obtained by crossing between Drosophila melanogaster and UAS-miR-981 by Tub-Ga180ts/ Tub-Ga14 system. The expression of Diptericin in the highly expressed Drosophila melanogaster miR-981 showed a significant decrease. However, there was no significant difference in the expression of other antimicrobial peptides such as Cecropin and Defensin. These results suggest that miR-981 may be directly involved in regulating the expression of the antimicrobial peptide Diptericin to regulate the immune response of Drosophila IMD signal. In order to further verify the interaction between miR-981 and Diptericin, the target information between Dipteri in and miR-981 was predicted by TargetScan and miRanda bioinformatics prediction software. The predicted results showed that there was a direct target action relationship between diptericin and miR-981. In order to confirm the relationship between miR-981 and DDiptericin, we used double luciferase report system in vitro to further study the expression of Diptericin in S2 cells of Drosophila melanogaster. The results showed that miR-981 could inhibit the expression of Diptericin. The inhibitory effect of miR-981 on Diptericin did not exist when we mutated the target sites between them, which indicated that miR-981 directly inhibited the expression of Dipterici. Subsequently, we reduced the expression of miR-981 to normal level in drosophila melanogaster. After the expression of miR-981 was decreased, the results of re-detection showed that there was no significant difference in the expression of diptericin between the control group and the control group. To sum up, we used biological experimental methods and bioinformatics analysis methods to prove that miR-981 can regulate the IMD immune signal pathway of Drosophila melanogaster by directly targeting Diptericin's 3G UTR sequence and then regulating the IMD immune signal pathway of Drosophila melanogaster (Drosophila melanogaster). This study provides a reference for the study of miRNA involved in the immune regulation of Drosophila melanogaster.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392;R-332
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