miR-981在果蠅Imd免疫響應(yīng)中的調(diào)控作用研究
發(fā)布時(shí)間：2018-07-18 08:56
【摘要】：黑腹果蠅作為重要的生物學(xué)研究模式動(dòng)物之一,在研究基因表達(dá)調(diào)控、神經(jīng)科學(xué)、人類疾病及遺傳機(jī)制等多個(gè)研究領(lǐng)域發(fā)揮出了重要作用。目前,有關(guān)果蠅的先天性免疫方面的研究已經(jīng)取得了諸多的成就,主要包括體液免疫和細(xì)胞免疫,其中Toll和Imd是果蠅最重要的體液免疫方式,并在近些年的研究過(guò)程中鑒定出了很多參與免疫響應(yīng)的基因。MicroRNA (miRNA)是非編碼小RNA家族的一員,長(zhǎng)度約為21-24nt的單鏈小分子RNA。miRNA可以通過(guò)靶向一個(gè)或多個(gè)靶基因,并且抑制靶基因的翻譯水平來(lái)調(diào)節(jié)基因的表達(dá)。而在果蠅體內(nèi),miRNA是否會(huì)通過(guò)與免疫基因相互作用來(lái)調(diào)節(jié)免疫響應(yīng),一直是我們感興趣的問(wèn)題。目前,只有極少的miRNA被發(fā)現(xiàn)參與調(diào)控果蠅的免疫應(yīng)答。本論文主要研究的是miR-981對(duì)于果蠅Imd免疫信號(hào)通路的調(diào)控機(jī)制,所采取的實(shí)驗(yàn)研究和所得結(jié)果如下:1.通過(guò)革蘭氏陰性菌刺激野生型果蠅,然后利用qRT-PCR技術(shù)檢測(cè)3h、6h、12h、24h、48h和72h果蠅體內(nèi)的miR-981和Imd信號(hào)通路的代表性抗菌肽Diptericin的表達(dá)量。結(jié)果發(fā)現(xiàn),在抗菌肽Diptericin的表達(dá)量先由上升隨后逐漸降低的整個(gè)應(yīng)答過(guò)程中,miR-981的表達(dá)量在6h和12h表現(xiàn)出顯著的升高,這些結(jié)果暗示果蠅Imd信號(hào)的激活引起了 miR-981表達(dá)量的變化。2.利用Tub-Ga180ts/+;Tub-Ga14系統(tǒng)果蠅與UAS-miR-981果蠅雜交,得到miR-981高表達(dá)的果蠅株。通過(guò)免疫損傷實(shí)驗(yàn)發(fā)現(xiàn),在miR-981高表達(dá)果蠅體內(nèi),Diptericin的表達(dá)量表現(xiàn)出顯著的下降。然而Attain、Cecropin和Defensin等其他抗菌肽的表達(dá)量卻沒(méi)有明顯的差異,這些結(jié)果暗示miR-981可能直接參與調(diào)控抗菌肽Diptericin的表達(dá)來(lái)調(diào)節(jié)果蠅Imd信號(hào)響應(yīng)的免疫應(yīng)答。3.為了進(jìn)一步驗(yàn)證miR-981和Diptericin之間的作用關(guān)系,我們先通過(guò)TargetScan和miRanda兩種生物信息學(xué)預(yù)測(cè)軟件預(yù)測(cè)了Dipteri in和miR-981之間的靶位點(diǎn)信息。預(yù)測(cè)結(jié)果均表明,miR-981和Diptericin存在著直接的靶作用關(guān)系。4.為了證實(shí)miR-981和DDiptericin的作用關(guān)系,我們?cè)隗w外采用雙熒光素酶報(bào)告系統(tǒng)在果蠅S2細(xì)胞內(nèi)進(jìn)行進(jìn)一步研究,結(jié)果表明miR-981確實(shí)能夠抑制Diptericin的表達(dá),而當(dāng)我們將二者之間的作用靶位點(diǎn)進(jìn)行突變后,miR-981對(duì)Diptericin的抑制作用就不存在了,這表明miR-981直接抑制了Dipterici 的表達(dá)。5.隨后,我們?cè)隗w內(nèi)將miR-981高表達(dá)果蠅體內(nèi)的miR-981表達(dá)量降至正常水平。在miR-981的表達(dá)量降低后,再次檢測(cè)的結(jié)果顯示,Diptericin的表達(dá)量與對(duì)照組相比沒(méi)有顯著的差別。綜上所述,我們采用生物學(xué)的實(shí)驗(yàn)研究手段以及生物信息學(xué)的分析方法,研究證明了黑腹果蠅miR-981可以通過(guò)直接靶向Diptericin的3'UTR序列進(jìn)而調(diào)控果蠅的Imd免疫信號(hào)通路。本論文的研究為探討miRNA參與果蠅免疫調(diào)控提供了參考。
[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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392;R-332
本文編號(hào)：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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392;R-332
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