【作者】 林强；

【导师】 程郢；

【作者基本信息】 云南大学 ， 发育生物学， 2024， 硕士

【摘要】 睡眠是动物界普遍的一种行为活动,无论是神经系统极为简单的软体动物还是神经系统极为复杂的高等灵长类动物都会有睡眠行为。睡眠障碍会对个体发育、行为、认知及寿命方面产生不同程度的影响。一个健康正常的睡眠被认为是个体健康的基础保障。在之前果蝇的研究中就已经发现果蝇与人的睡眠与觉醒周期相似,在果蝇的睡眠行为表现,药物反应、基因表达等方面也表现出与人类睡眠极大的相似性。研究表明,usp5的缺失会导致果蝇光感受器发育受损,可能影响果蝇对于光刺激的反应而调节果蝇的睡眠觉醒。usp52与usp5在生物体内都能拮抗性的调节mTOR信号通路。本研究利用果蝇作为模式生物研究usp52与usp5缺失对于果蝇睡眠的影响。本研究通过CRSIPR/Cas9技术,针对usp52和usp5两个基因进行KO型果蝇品系的建系,两个基因分别设计了 23个和5个基因敲除靶点,以果蝇的显微注射进行靶点筛选,最后得到usp52(5号、12号)及usp5(2号、3号)的有效靶点能够导致果蝇碱基缺失,并证实其突变位点均为非三的整数倍缺失,非三整数倍缺失会导致移码突变从而影响基因表达。将建立好的单靶点KO果蝇品系分别命名为usp525,usp52p12和usp52,usp53。在建立好usp52KO型和usp5KO 型果蝇品系后,分别将两个基因的两个的靶点杂交起来,得到双靶点KO果蝇(usp525/12和usp52/3)作为实验组,以w1118的野生型果蝇作为对照组,进行睡眠实验。与对照组相比usp525/12和usp52/3的果蝇无论雌雄都出现了睡眠时间延长的嗜睡现象。通过大脑组织免疫荧光结果发现usp52基因在果蝇大脑内集中表达在视叶与中央脑交界处。通过RNA-seq结果分析,其中我们发现usp525/12KO型果蝇与野生型果蝇之间有显著的转录水平差异变化,我们发现其中有342个差异表达上调基因,有415个差异表达下调基因。通过GO生物过程功能富集,我们发现usp525/12KO型果蝇与野生型果蝇相比明显对光刺激敏感度降低。usp52的缺失会造成果蝇睡眠时间延长,从RNA-seq测序结果可知,睡眠时间延长的原因可能是usp52 KO型果蝇对光刺激的敏感度下降。结合组织免疫荧光实验结果可知,usp52KO型果蝇与w1118的果蝇相比视神经叶与中央脑交界处缺失Usp52,所以usp52KO型果蝇光敏感度下降可能光信号无法传递到中央脑处理相关。提示果蝇体内Usp52参与光信号通路相关过程。已有研究表明Usp5的缺失会导致果蝇光源感受器发育受损,本次研究发现usp5的缺失会导致果蝇出现睡眠延长的嗜睡现象。可能的原因是光感受器受损后导致usp5 KO型果蝇对光刺激敏感度降低。表明这两个基因对于果蝇的睡眠觉醒具有重要作用。更多还原

【Abstract】 Sleep is a common behavioral activity in the animal kingdom,both in mollusks with extremely simple nervous systems and in higher primates with extremely complex divine systems.Sleep disorders can have varying degrees of impact on individual development,behavior,cognition,and lifespan.A healthy and normal sleep is considered a fundamental guarantee of health.In previous studies on fruit flies,it has been found that their sleep and awakening cycles are similar to those of humans.They also exhibit significant similarities in their sleep behavior,drug responses,gene expression,and other aspects to human sleep.Research has shown that the absence of usp5 can lead to impaired development of light source receptors in fruit flies,and the response to light source stimulation regulates their sleep awakening.There are also studies indicating that both usp52 and usp5 can regulate the mTOR signaling pathway in living organisms.This study used Drosophila as a model organism to investigate the effects of usp52 and usp5 deficiency on Drosophila sleep.This study used CRSIPR/Cas9 technology to establish KO type Drosophila strains targeting the usp52 and usp5 genes.23 and 5 gene knockout targets were designed for each gene,and target screening was performed using microinjection of Drosophila.Finally,effective targets for usp52(5,12)and usp5(2,3)were found to cause base deletions in Drosophila,and it was confirmed that their mutation sites were all non triple multiples deletions,which could lead to frameshift mutations and affect gene expression.Name the established single target KO fruit fly strains usp525,usp5212,and usp52,usp23,respectively.After establishing the usp52KO and usp5KO Drosophila strains,two target genes of the two genes were hybridized to obtain dual target KO Drosophila(usp525/12 and usp5213)as the experimental group,and wild-type Drosophila w1118 as the control group for sleep experiments.Compared with the control group,both male and female fruit flies with usp525/12 and usp522+3 exhibited prolonged sleep duration and drowsiness.Through immunofluorescence analysis of brain tissue,it was found that the usp52 gene is concentrated in the Drosophila brain at the junction of the optic lobe and the central brain.Through RNA seq analysis,we found significant transcriptional differences between KO type Drosophila and wild-type Drosophila.We found 342 differentially upregulated genes and 415 differentially downregulated genes.Through GO biological process functional enrichment,we found that the usp52KO type Drosophila showed a significant decrease in sensitivity to light stimulation compared to the wild type Drosophila.The absence of usp52 can cause prolonged sleep time in fruit flies.According to RNA seq sequencing results,the reason for prolonged sleep time may be the decreased sensitivity of usp52 KO type fruit flies to light stimulation.Based on the results of the woven immunofluorescence experiment,it can be concluded that the usp52KO type fruit fly lacks USP52 at the junction of the optic nerve lobe and the central brain compared to the W1118 fruit fly.Therefore,the decreased photosensitivity of the usp52KO type fruit fly may prevent the transmission of light signals to the central brain for processing.Previous studies have shown that the absence of Usp5 can impair the development of light source receptors in fruit flies.This study found that the absence of Usp5 can lead to prolonged sleep and drowsiness in fruit flies.The possible reason is that damage to the photoreceptors leads to a decrease in the sensitivity of usp5 KO type fruit flies to light stimulation.It is suggested that Usp52 is involved in processes related to the light signaling pathway in fruit flies.Although the absence of Usp52 and Usp5 leads to prolonged sleep time in fruit flies,the sites of their action may be different.Usp5 affects fruit flies’ reception of light sources from the receiving light source(photoreceptor),while Usp52 affects the transmission of light source signals to the central brain.However,the loss of both leads to drowsiness in the sleep phenotype of fruit flies.This also indicates that these two genes play an important role in the sleep awakening of fruit flies.更多还原