Preliminary Identification Of Gene Localization And Function Of Nanos Protein In Schistosoma Japonicum

Objective Nanos is a Zn-finger domain, RNA binding protein acting as a translational repressor. It is known to act in concert with its canonical partner Pumilio. Together, these proteins repress translation of their target genes. Nanos was first described as a posterior marker in Drosophila and subsequent studies established its main role in Reproduction organs.Nanos can regulate the update of germ cell and migration to gonade,inhibit early maturing and tendency of somatic cells.the In this study,we initially identified the location and function of Nanos protein by in situ hybridization and RNAi.Method We used the software BLAST to select a specific sequence of Nanos gene open reading frame(ORF) preparing for the probe needed in situ hybridization.This specific sequence was called Nanos’.Nanos’ sequence was amplified via PCR and connected with PMD18-T plasmid,then sequencing was carried out after enzyme identification.After sequencing correctly,Nanos’ sequence was obtained by double enzyme digestion and connected with pspt19 plasmid,then sequencing was carried out after enzyme identification.Linearize the extracted plasmids with restrictive nucleic acid enzymes(ecor1 or hind3)and check for complete linearization by running a sample on an electrophoresis gel.The RNA probes can be synthesized by transcription in vitro using the DIG RNA Labeling Kit(SP6/T7)according to the detailed instruction manual.Labeled probe are stable for a minimum of one year when store at-80℃.The final concentration of the labeled probe used for in situ hybridization was 5ng/ml,and results reviewed that Nanos expressed in the ovary and vitellarium,however,male worm showing no positive signals.We assume that Nanos expression levels of male worms may be below the detection thresholds of the in-situ hybridisation protocol.In this study,RNA interference(RNAi) approaches were used to investigate the effects of knockdown of the schistosoma japonicum Nanos gene.Five different short interfering RNAs targeting different regions of the gene were designed to suppress the expression of Nanos in Schistosoma japonicum using a transfection method.Mice were infected through percutaneous exposure to cercariae and schistosomula were harvested 18 days post infection.These schistosomula were cultured in complete RPMI 1640 medium containing 10 % serum(Gibco), at 37 °C in 5 % CO2 in twelve-well culture plates.Schistosomula were treated by transfected with N1, N2,N3, N4,N5,or nonspecific si RNA for 48 h, at a 200-n M final concentration.Silence effects were detected by real-time PCR, Western blot analysis, and confocal laser scanning microscopy.18 days of Schistosoma japonicum infected with the retrovirus, the experimental group could express Nanos gene sh RNA retrovirus, control group expression disorder not for Schistosoma japonicum any gene sh RNA retrovirus. 14 days after virus infection, total RNA was extracted from the untreated group, control group and experimental group, through the Q-PCR experiment control group showed that Nanos gene expression level is not reduced, the level of experimental group Nanos gene expression decreased about 65%. Egg counting results showed that the experimental group compared with control group, the egg reduction rate was 28%, the experimental group were significantly smaller and lower maturity. Morphological changes of the male and female worms were observed after interference under confocal laser microscopy, of which around the female ovarian large vacuoles, ovarian cells were scattered, abnormal shape; and there is no obvious morphological changes in testis of male.Results The results of whole mount in situ hybridization for 18-day-old,24-day-old and 42-day-old schistosoma japonicum reveal that Nanos gene express in ovary and vitellaria and no signals were observed in male worms.The results of RNAi showed that Nanos gene expression level decreased about 65%, egg count showed egg reduction rate of 28%, eggs significantly smaller and maturity decreased significantly. Laser confocal microscopy showed large vacuoles around the female ovary, ovarian cells were loose, abnormal shape; and there is no obvious morphological changes in testis of male.