摘要
目的研究日本血吸虫Mago nashi蛋白编码基因在虫体生殖器官发育方面的功能。方法体外PCR法合成Mago nashi双链RNA(dsRNA),将SjMago nashi基因dsRNA产物和阴性对照lacZ基因dsRNA产物分别电击转染至机械脱尾的日本血吸虫童虫体内,将部分电击转染的童虫作体外培养,在培养后第1、3和5天以TRIzol法同时提取其总RNA和总蛋白。实时荧光定量RT-PCR和蛋白质印迹(Western blotting)分别检测Mago nashi基因和蛋白表达产物。电击转染的童虫注射入6只BALB/c小鼠体内,每鼠约1 000条,6周后取出虫体,盐酸卡红染色,在激光共聚焦显微镜下观察虫体内部各器官形态特征,测量虫体相关指标。结果在电击转染后的第1、3和5天,SjMago nashi dsRNA组目的基因的转录水平较阴性对照组分别下降了22%、69%和80%,蛋白质水平较阴性对照组分别下降了12%、39%和56%。激光共聚焦显微镜观察发现,SjMago nashi dsRNA组中的雄虫睾丸内有较多精子,呈泛雄性化表现,而雌虫的卵巢和卵黄腺无明显特征变化。与阴性对照组相比,SjMago nashi dsRNA组的雄虫的体宽、睾丸长和睾丸宽,雌虫的体宽、卵巢长和卵巢宽均明显缩小(P<0.05)。结论 SjMago nashi dsRNA可特异性抑制日本血吸虫靶基因及其编码蛋白的表达,SjMago nashi基因为日本血吸虫生殖相关基因,在生殖系统器官的正常发育中起一定作用。
Objective To study the function of Mago nashi gene in reproductive system of Schistosoma japonicum.Methods dsRNA products of SjMago nashi gene and control gene（lacZ） were generated by in vitro transcription.SjMago nashi dsRNA and control（lacZ） dsRNA were electroporated into mechanically transformed schistosomula.Aliquots of parasites（1 000） were harvested at day 1,3,and 5 after electroporation,respectively.Total RNA and proteins were isolated simultaneously using TRIzol reagent.Levels of SjMago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis,respectively.About 1 000 dsRNA-electroporated schistosomula were injected into each BALB/c female mouse.Six weeks later the worms were collected,fixed,stained,clarified,dehydrated and mounted.The male and female reproductive organs were observed and measured under the confocal laser scanning microscope.Results At day 1,3 and 5 post-electroporation,22%,69%,and 80% reduction in Mago nashi mRNA levels were detected respectively in SjMago nashi dsRNA-electroporated schistosomula（experiment group） compared to parasites treated with control dsRNA（control group）;and schistosomula of experiment group exhibited 12%,39%,and 56% decreased in Mago nashi protein expression levels in comparison to the control group,respectively.In experiment group there were many spermatozoa in testicular lobes and no changes were observed in ovary and vitelline gland.Compared to control group,adult worms in experiment group were smaller in the body width,the width and length of testicles and ovaries（P0.05）.Conclusion Mago nashi dsRNA can specifically inhibit the expression of target gene and protein.SjMago nashi gene is a reproduction-related gene.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2011年第2期87-92,共6页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.30771882)
安徽省教育厅自然科学基金重点项目(No.2005KJ045ZD)~~
