家蝇MdSirt1和MdSirt4基因的克隆及功能分析

发布时间：2018-04-09 02:21

本文选题：家蝇　切入点：MdSirt1　出处：《河北大学》2016年硕士论文

【摘要】：随着对Sirtuins家族蛋白研究的不断深入,关于其功能的报道也越来越广泛。通过转录组和数字表达谱分析,我们发现家蝇在受到细菌刺激后体内Sirt1和Sirt4基因表达水平发生显著变化,提示它们可能参与家蝇免疫应答过程。本文基于此,本工作以此为出发点对家蝇应激过程中Sirt1和Sirt4基因表达的变化规律及其可能存在的调控机制进行了研究。主要内容包括:1、克隆了家蝇Sirt1和Sirt4基因c DNA序列,分别命名为Md Sirt1和Md Sirt4,并对其进行了生物信息学分析。两条基因所编码的蛋白Md SIRT和Md SIRT4各具有一段保守的SIR2保守结构域,两者氨基酸序列与其它物种中的同源蛋白之间均具有较高的同源性。2、利用实时荧光定量PCR和蛋白免疫印迹方法对Md Sirt1和Md Sirt4在家蝇不同发育时期、不同组织和应激过程中的表达量变化进行了分析。Md Sirt1和Md Sirt4在家蝇受到细菌刺激时分别上调和下调,同时检测到Md SIRT4在蛋白水平也发生了相似的变化。3、Md Sirt1和Md Sirt4在家蝇受到热激处理时m RNA表达量分别上调和下调,同时检测到Md SIRT4在蛋白水平也发生了相似的变化。4、Md Sirt1和Md Sirt4在家蝇受到重金属影响后m RNA表达量均上调,同时检测到Md SIRT4在蛋白水平也发生了相似的变化。5、在对Md Sirt1干扰后我们发现家蝇幼虫应对细菌刺激的能力下降;而对Md Sirt4进行了干扰后,我们发现家蝇核转录因子Md NF-κB表达量上升,同时检测到家蝇抗菌肽的表达量上调。
[Abstract]:With the further study of Sirtuins family proteins, more and more reports about its function have been reported.By the analysis of transcriptome and digital expression profile, we found that the expression level of Sirt1 and Sirt4 genes in housefly was significantly changed after bacterial stimulation, suggesting that they may be involved in the immune response of housefly.Based on this work, the changes of Sirt1 and Sirt4 gene expression and their possible regulatory mechanisms during housefly stress were studied.The main contents include: 1, cloned the c DNA sequence of housefly Sirt1 and Sirt4 gene, named Md Sirt1 and Md Sirt4 respectively, and analyzed them by bioinformatics.The proteins Md SIRT and Md SIRT4 encoded by the two genes each have a conserved SIR2 conserved domain.The amino acid sequence of Md Sirt1 and Md Sirt4 in Musca domestica at different developmental stages were analyzed by real-time fluorescence quantitative PCR and Western blot.The changes of expression of Md Sirt1 and Md Sirt4 in different tissues and stress were analyzed. Md Sirt1 and Md Sirt4 were up-regulated and down-regulated respectively when housefly was stimulated by bacteria.It was also found that Md SIRT4 had a similar change in protein level. Md Sirt1 and Md Sirt4 were up-regulated and down-regulated respectively during heat shock treatment of Musca domestica.It was also found that Md SIRT4 had a similar change in protein level. 4Md Sirt1 and Md Sirt4 were up-regulated after heavy metal exposure in Musca domestica.At the same time, Md SIRT4 had a similar change in protein level. 5. After interfering with Md Sirt1, we found that the ability of housefly larvae to respond to bacterial stimulation was decreased, while Md Sirt4 was interfered with.We found that the expression of nuclear transcription factor Md NF- 魏 B was increased and the expression of antimicrobial peptide in housefly was up-regulated.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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