| Objective: Nanos is RNA binding protein containing the structure of zinc refers.It can be in interaction with Pumilio protein and plays an important role in transcription of survival-related genes of primordial germ cells.Nanos can regulate the transcription and translation of gene and the location of mRNA in time and space,which is crucial in starting the post-polarization development of Drosophila embryo.Nanos is also a necessary factor in the migration and differentiation of primordial germ cells as well as for their fate maintenance of all animals tested including Caenorhabditis elegans,Danio rerio,Bombyx mori,Xenopus,and mouse.It is closely associated with the development of genitalia in a wide range of species.We questioned whether Nanos in Schistosoma japonicum(SjNanos)was involved in the reproductive organs development of Schistosoma japonicum(S.japonicum).Methods: Firstly,similarity analysis of Nanos protein was performed using BLAST in NCBI and DNAMAN software to study the Nanos homology.Then,in situ hybridization was used to observe the location of Nanos gene in S.japonicum.Subsequently,the paired schistosomes of 28 days post infection were transfected with specific SjNanos siRNA three times,and cultured in vitro for 10 days.The knockdown effects were measured in mRNA and protein levels by Quantitative PCR(Q-PCR)and Western bolt.We observed the morphological changes of reproductive organs of S.japonicum by Confocal Laser Scanning Microscopy(CLSM)and the egg production of S.japonicum using light microscopy after SjNanos gene silence.Subsequently,the expression of those genes,which may interact with SjNanos1 gene and be related to egg formation,were also accessed using Q-PCR after silencing SjNanos gene.Results: As the results of protein similarity analysis showing,SjNanos1 presented43% identity with Nanos2,but only 25% identity with Nanos1 in S.mansoni.AsNanos2 is mainly expressed in vitellarium of S.mansoni,it may be related to reproduction development.Therefore,SjNanos1 function was researched in S.japonicum reproduction.By in situ hybridization,we found that SjNanos1 gene was expressed mainly in reproductive organs of S.japonicum.We confirmed that SjNanos1 expression in the mRNA and protein levels were suppressed compared to that of the controls after SjNanos1 gene knockdown.As the results of CLSM showing,after RNA interference(RNAi),in the males,the size of the testicular lobes was mildly shrunk,and nearly no mature elongated sperms were observed in the ventral part of the lobes but some immature spermatogonia.Besides,obvious cracks were found in each testicular lobe.In the females,the number of mature oocytes in the ovary was decreased obviously,and the number of immature oocytes was increased compared to the controls.SjNanos gene knockdown also suppressed the maturation of vitellaria,and induced vitelline cells to shrink.Moreover,silencing SjNanos1 gene also induced the reduction of the egg.Compared to the mock group,the number of egg,on the fourth,seventh and tenth day,reduced by approximately 41%,56% and 71%,respectively,and the number of egg in female uterus also reduced by approximately 50% on tenth day.In addition,SjNanos1 gene silence affected the changes of these genes interacting with SjNanos1 gene and related to egg formation,including Pumilio,CNOT6 L,and Fs800.After SjNanos1 siRNA3 treated,the expression level of Pumilio gene increased by approximately three fold.The expression level of CNOT6 L reduced by approximately50% at this point.Notably,the expression level of Fs800 gene transcripts in S.japonicum decreased significantly by approximately 90%.Conclusion: By silencing the expression of Sj Nanos1,the morphology of reproductive organs of parasite,especially the female ovaries,vitellarium and the male testes,is changed obviously,and the formation and production of egg is also affected significantly.Therefore,we determine that SjNanos1 gene plays an important role in the reproductive development and the egg production in S.japonicum. |
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