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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 protein encoding gene of Schistosoma japonicum in development of reproductive organs of parasites. Methods Mago nashi double-stranded RNA (dsRNA) was synthesized by in vitro PCR. The dsRNA products of SjMago nashi gene and the negative control lacZ gene were transfected into mechanically de-stained Schistosoma japonicum by electric shock respectively. For in vitro culture, total RNA and total protein were simultaneously extracted by TRIzol method on the 1st, 3rd and 5th day after culture. Real-time fluorescent quantitative RT-PCR and Western blotting were used to detect the expression of Mago nashi gene and protein. The electroporated mice were injected into 6 BALB / c mice, about 1 000 in each mouse. After 6 weeks, the parasites were removed and stained with carbachol hydrochloride. The morphological features of various organs in the parasites were observed under laser scanning confocal microscope , Measurement of parasites related indicators. Results The transcription level of SjMago nashi dsRNA group decreased by 22%, 69% and 80%, respectively, on the 1st, 3rd and 5th day after electroporation. Compared with the negative control group, the protein level decreased by 12% %, 39% and 56%. Confocal laser scanning microscopy showed that there were more spermatozoa in the testis of the male SjMago nashi dsRNA group, showing a pan-male performance, while the female ovary and the yolk glands showed no obvious changes. Compared with the negative control group, the body width, testicular length and testicular width, body width, ovarian length and ovary width of male SjMago nashi dsRNA group were significantly reduced (P <0.05). Conclusion SjMago nashi dsRNA can specifically inhibit the expression of the target gene and its encoded protein of Schistosoma japonicum. The SjMago nashi gene is a reproductive related gene of Schistosoma japonicum, and plays a role in the normal development of reproductive organs.