日本血吸虫Vasa3基因功能的初步研究

发布时间：2018-04-24 08:28

本文选题：日本血吸虫 + 生殖发育　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的:Vasa基因首次在果蝇中发现,是胚胎形成和生殖细胞发育过程中所必需的因子。在斑马鱼中,Vasa基因作为一个母源性基因产物,主要存储于卵母细胞的胞质中,是生殖细胞形成的关键因素。由于Vasa基因与大多数物种生殖器官的生长发育密切相关,因此我们推测日本血吸虫Vasa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:经过整虫原位杂交的方法对Vasa3基因在日本血吸虫18天、24天、36天和42天不同发育阶段成虫进行基因定位;再通过RNAi的方法鉴定SjVasa3基因的功能。以SjVasa3为靶序列,根据siRNA筛选原则通过软件设计出3个不同靶区域的SjVasa3 siRNAl,SjVasa3 siRNA2和SjVasa3siRNA3,经过荧光定量PCR确定有效siRNA,再利用该siRNA分别3次体外转染28天的日本血吸虫成虫。RNA干扰10天后首先通过荧光定量PCR和Western Blot方法确定其基因和蛋白水平消减效果,再通过激光共聚焦显微镜观察雌虫的卵巢、卵黄腺,雄虫的睾丸、贮精囊内的精子和子宫内虫卵的数量。另外,分别在RNA干扰的第4、第7和第10天通过光学显微镜下观察虫体合抱情况、虫卵形态和数量的变化。结果:本研究课题前期通过整虫原位杂交的方法对不同发育阶段的日本血吸虫成虫进行定位,发现在18天感染的日本血吸虫的睾丸和卵巢没有明显的阳性信号。但是在24天、36天和42天感染的日本血吸虫,SjVasa3在雌虫卵巢的后端有所表达,而在雄虫的睾丸中没有出现明显的阳性信号。荧光定量PCR结果显示得出有效靶序列为SjVasa3siRNAl。该siRNA体外转染3次的虫体培养10天后,与错乱siRNA处理组相比,SjVasa3在转录水平下降了 80%,蛋白水平下降了 60%。激光共聚焦扫描显微镜的观察结果显示,消减了SjVasa3基因,在日本血吸虫雄虫的睾丸萎缩、贮精囊内的精子变少变圆;雌虫卵巢内成熟卵母细胞和不成熟卵母细胞比例分布失调,卵黄腺内的卵黄细胞排列顺序发生紊乱;子宫内虫卵的数量明显减少。在SjVasa3基因消减后,通过光学显微镜进行虫卵计数,与错乱siRNA处理组相比,发现在第4天和第7天雌虫的产卵数量和子宫内虫卵的数量变化都不具有统计学差异,在第10天时雌虫的产卵数量下降约60%和子宫内虫卵的数量下降约68% (p0.05)。同时虫体的合抱率在第7天和第10天时分别下降了约30%和47% (p0.05),在第4天虫体的合抱率不具有统计学差异。结论:通过RNAi技术,体外验证消减日本血吸虫Vasa3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,证明了日本血吸虫Vasa3基因在其生殖系统器官发育和虫卵形成中具有重要的功能。
[Abstract]:Objective to identify for the first time in Drosophila the Vasa gene as an essential factor in embryogenesis and germ cell development. As a mother gene product, Vasa gene is mainly stored in the cytoplasm of oocytes and is the key factor of germ cell formation in zebrafish. Because the Vasa gene is closely related to the growth and development of reproductive organs in most species, we speculate that the protein encoded by Vasa gene of Schistosoma japonicum also plays a role in the development of reproductive organs of Schistosoma japonicum. Methods: by in situ hybridization, the Vasa3 gene was located in 18 days, 24 days, 36 days and 42 days, and the function of SjVasa3 gene was identified by RNAi. Using SjVasa3 as the target sequence, According to siRNA screening principle, three different target regions of SjVasa3 siRNAlSjVasa3 siRNA2 and SjVasa3 siRNA3 were designed by software. The effective siRNAs were determined by fluorescent quantitative PCR. The siRNAs were transfected into Schistosoma japonicum adult for 28 days in vitro three times with the siRNA for 10 days. Fluorescence quantitative PCR and Western Blot were used to determine the subtractive effect of the gene and protein levels. The number of ovaries, yolk glands, testis of males, seminal vesicles and the number of eggs in uterus were observed by confocal laser microscopy. In addition, on the 4th, 7th and 10th day of RNA interference, the morphology and number of eggs were observed under optical microscope. Results: in the early stage of this study, the adult Schistosoma japonicum in different developmental stages was located by in situ hybridization. It was found that there were no obvious positive signals in testis and ovaries of Schistosoma japonicum infected at 18 days. However, Sj Vasa3 of Schistosoma japonicum infected at day 24 and day 42 was expressed at the posterior end of the female ovary, but there was no obvious positive signal in the testis of the male worm. Fluorescence quantitative PCR showed that the effective target sequence was SjVasa3siRNAl. After 10 days of culture of the siRNA transfected for three times in vitro, Sj Vasa3 decreased by 80% at the transcription level and the protein level decreased by 60% compared with that of the siRNA treatment group. The results of confocal laser scanning microscopy showed that the SjVasa3 gene was subtracted, the testis of Schistosoma japonicum shrank, the spermatozoa in the seminal vesicle became less round, and the proportion of mature oocytes and immature oocytes in the ovaries of female worms was misdistributed. The order of egg yolk cells in yolk gland was disordered, and the number of eggs in uterus decreased obviously. After subtractive of SjVasa3 gene, the egg counts were counted by optical microscope. The results showed that there was no significant difference in the number of eggs laid by females and the number of eggs in uterus on the 4th and 7th day after treatment with siRNA. On the 10th day, the number of eggs laid by females decreased by about 60%, and the number of eggs in uterus decreased by about 68% (p0.05). At the same time, the rate of cuddling decreased by about 30% and 47% respectively on the 7th and 10th day, but there was no significant difference on the 4th day. Conclusion: the expression of Vasa3 gene of Schistosoma japonicum was subtracted by RNAi technique in vitro. The morphology of female ovaries, yolk glands and testis of males were significantly changed, and the number of eggs laid by females was also significantly affected. It is proved that the Vasa3 gene of Schistosoma japonicum plays an important role in the development of reproductive organs and egg formation of Schistosoma japonicum.
【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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