Nanos1基因在日本血吸虫生殖器官中的功能研究

发布时间：2018-07-28 11:15

【摘要】：目的:Nanos基因编码一种含锌指结构的RNA结合蛋白,可以与其起协同作用的Pumilio蛋白结合,在原始生殖细胞生存相关基因的转录调控中起到重要作用。Nanos蛋白在时间、空间上对基因转录翻译和m RNA定位的调控,是启动果蝇胚胎后极化发育过程的关键机制。Nanos基因在原始生殖细胞的迁移和分化过程中发挥重要的功能,并且在秀丽隐杆线虫、斑马鱼、家蚕、非洲蟾蜍和小鼠等测试动物的生长发育方面也起到必不可少的作用。由于Nanos基因与大多数物种生殖器官的发育密切相关,因此我们推测日本血吸虫Nanos基因编码的蛋白在其生殖器官的发育中也起一定的作用。方法:首先,使用DNAMAN软件和NCBI中BLAST,进行Nanos蛋白的同源性分析,来确定日本血吸虫Nanos的分型;然后再通过原位杂交的方法,探究Nanos基因在日本血吸虫的定位情况;随后,用针对Nanos基因的si RNA转染感染后28天合抱的日本血吸虫,体外培养10天,期间每隔3天转染一次si RNA,共转染三次;使用TRIzol方法提取虫体总RNA和总蛋白,通过荧光定量PCR以及Western blot方法分析其在m RNA和蛋白水平的消减情况;Nanos基因干扰后,使用盐酸胭脂红染色虫体的方法,通过激光共聚焦扫描显微镜观察日本血吸虫生殖器官的形态改变;我们还使用普通光学显微镜观察Nanos基因的干扰对日本血吸虫虫卵数量的影响;最后,在Nanos基因消减后,通过荧光定量PCR方法检测可能与Nanos基因相互作用以及虫卵形成相关基因表达水平的变化。结果:Nanos蛋白同源性分析结果显示,日本血吸虫Nanos1(SjNanos1)蛋白与曼氏血吸虫Nanos2蛋白同源性为43%,与曼氏血吸虫Nanos1蛋白同源性为25%。依据文献报道,曼氏血吸虫Nanos2主要表达在卵黄腺中,可能与生殖系统发育相关,故将SjNanos1基因作为本课题的研究对象;原位杂交的结果显示,SjNanos1基因主要表达在合抱后日本血吸虫雌虫和雄虫生殖器官中,而合抱前的日本血吸虫并未发现SjNanos1基因的表达;SjNanos1基因的si RNA干扰后,与空白组相比,SjNanos1的表达在m RNA和蛋白质水平均显著降低;激光共聚焦扫描显微镜的观察结果显示,SjNanos1基因消减后,在日本血吸虫雄虫中,睾丸轻微收缩,并且在睾丸的腹侧部分几乎没有观察到成熟的细长的精子;同时,在每个睾丸中都发现有明显的裂隙。对于血吸虫雌虫,其卵巢中成熟卵母细胞减少,不成熟卵母细胞增多,卵黄腺中成熟的卵黄细胞也明显减少并且卵黄细胞也明显萎缩;在SjNanos1基因消减后,通过普通光学显微镜进行虫卵计数,发现雌虫的产卵数量在第4、第7天和第10天时分别下降了大约41%、56%和71%,并且雌虫子宫中虫卵的数量也明显减少,培养10天下降了50%左右;此外,SjNanos1基因沉默也影响了与SjNanos1基因相互作用的以及虫卵形成相关基因的变化,如Pumilio,CNOT6L和Fs800基因。在SjNanos1基因干扰后第10天,Pumilio基因的表达量增加了3倍左右,CNOT6L基因的表达水平下降了约50%。更值得注意的是,Fs800基因在日本血吸虫中的表达减少约90%。结论:通过干扰日本血吸虫SjNanos1基因的表达,血吸虫雌虫卵巢、卵黄腺和雄虫睾丸形态发生明显的变化,并且雌虫的产卵量也受到显著的影响,因此我们可以得出结论,SjNanos1基因在日本血吸虫生殖器官的发育以及虫卵的形成和产生中发挥重要的功能。
[Abstract]:Objective: the Nanos gene encodes a RNA binding protein containing the zinc finger structure, which can play an important role in the coordination of Pumilio protein, which plays an important role in the transcription regulation of the survival related genes of the primitive germ cells..Nanos protein is in time, and the regulation of gene translation and m RNA localization in space is to start the post polarization hair of the Drosophila melanogaster. .Nanos gene plays an important role in the migration and differentiation of primordial germ cells, and plays an essential role in the growth and development of Caenorhabditis elegans, zebrafish, silkworm, African toad and mice, as well as the development of the Nanos gene and the reproductive organs of most species. Therefore, we speculate that the protein encoded by the Nanos gene of Schistosoma japonicum also plays a role in the development of its reproductive organs. Methods: first, using DNAMAN software and BLAST in NCBI to analyze the homology of Nanos protein to determine the typing of Nanos in Schistosoma japonicum, and then to explore the Nanos gene by the method of in situ hybridization. In the location of Schistosoma japonicum, then transfected with Si RNA of Nanos gene for 28 days after infection, the Schistosoma japonicum was cultured for 10 days in vitro, and Si RNA was transfected once every 3 days and three times were transfected. TRIzol method was used to extract the total insect RNA and total protein, and the M RNA and Western blot methods were analyzed by fluorescence quantitative PCR and Western blot methods. After Nanos gene interference, the morphological changes of the reproductive organs of Schistosoma japonicum were observed by laser confocal scanning microscope with the method of carmine dyed with carmine. We also observed the effect of the interference of Nanos gene on the number of eggs of Schistosoma japonicum by ordinary optical microscope; finally, in the Nanos base After the reduction, the changes in the interaction of Nanos gene and the gene expression level of the egg formation were detected by the fluorescence quantitative PCR method. Results: the Nanos protein homology analysis showed that the homology of the Nanos1 (SjNanos1) protein of Schistosoma japonicum and the Nanos2 protein of Schistosoma mansoni was 43%, and the homology of the Nanos1 protein of Schistosoma mansoni. According to 25%., it is reported that the Nanos2 of Schistosoma mansoni is mainly expressed in the yolk gland and may be related to the development of the reproductive system. Therefore, the SjNanos1 gene is used as the subject of this study. The results of in situ hybridization show that the SjNanos1 gene is mainly expressed in the female and male reproductive organs of the Schistosoma japonicum after the clasp, and the Japanese blood sucking before the joint is held. The SjNanos1 gene was not found in the insect; the expression of SjNanos1 in the m RNA and protein levels decreased significantly after the Si RNA interference of the SjNanos1 gene. The results of the confocal laser scanning microscope showed that after the reduction of SjNanos1 gene, the testicles contracted slightly in the Japanese blood sucking males and the ventral side of the testicles. Part of the mature slender sperm was almost not observed; at the same time, obvious fissures were found in each testis. For schistosomiasis, the mature oocyte in the ovary decreased, the immature oocyte increased, the yolk cells in the yolk gland decreased obviously and the yolk cells atrophied obviously; the SjNanos1 gene subtracted. Then, the number of eggs was counted by ordinary optical microscopy, and the number of eggs dropped by about 41%, 56% and 71%, respectively, at fourth, seventh and tenth days, and the number of eggs in the womb of the female decreased significantly, and the culture decreased by about 50% in 10 days. In addition, the SjNanos1 gene silencing also affected the interaction with the SjNanos1 gene. Changes in the genes related to the formation of eggs, such as Pumilio, CNOT6L and Fs800 genes. The expression of Pumilio gene increased by about 3 times after the SjNanos1 gene interference, and the expression level of the CNOT6L gene decreased by about 50%.. The expression of Fs800 gene in Schistosoma japonicum was reduced by about 90%. conclusion: by interfering with the Japanese blood sucking. The expression of the SjNanos1 gene, the ovary of the female schistosomiasis, the yolk gland and the testis of the male, and the egg production of the female are also significantly affected. Therefore, we can conclude that the SjNanos1 gene plays an important role in the development of the reproductive organs of Schistosoma japonicum and the formation and production of the eggs.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R383.24

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