Identification And Characterization Of A Bone Morphogenetic Protein Homologue Of Schistosoma Japonicum

Background:Bone morphogenetic proteins (BMPs) show multifunctional in the regulation of cellular proliferation, survival, differentiation and apoptosis during the biological processes. Despite there has been a recognition that a BMP signaling pathway exists in schistosomes, the BMP homologue and its function in the parasitic platyhelminth Schistosoma japonicum remained unidentified, and what the role of this growth factor is in the embryonic patterning of s. japonicum also needs to be explored.Objectives:In this study, a bone morphogenetic protein (BMP) homologue of S. japonicum was determined to be cloned and characterized.Methods and results:The SjBMP mRNA cloned by rapid amplification of cDNA ends method (RACE) is3,020nt in full length and encodes a product of928amino acids that includes a TGF-β superfamily conserved domain at the C-terminal. BLAST analysis showed that SjBMP has68%,51%and43%identity at the amino acid level with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica, respectively. Real-time semiquantitative PCR detected that SjBMP is expressed in lung-stage schistosomula, liver-stage schistosomula, adult male and female worms and eggs. There were more SjBMP transcripts in lung-stage schistosomula (～27-fold) and eggs (～37-fold) than in other stages, and more in adult male worms than in the adult females. Immunohistochemical detection based on rabbit antisera against the recombinant SjBMP protein detected that SjBMP protein expressed and distributed generally in adult worms and eggs without obvious tissue specificity. After treatment with SjBMP double-stranded RNA (dsRNA) at a concentration of8×10-2μg/ml within one week, the level of SjBMP mRNA of the cultured adult mix-sex S. japonicum decreased at a range of～25%～98%(P<0.05) compared with the level of SjBMP mRNA in the blank control, and there was no significant difference (P>0.05) in SjBMP mRNA level between the EGFP dsRNA-treated negative control and the blank control. SjBMP dsRNA mediated RNAi in vitro led to less eggs and higher percent of abnormal eggs produced by the treated paired worms compared with the eggs laid by the blank control group on the2nd day, but no significant difference (P>0.05) was observed in both the egg count and the percent of abnormal eggs between the two groups on the7th day, becasue the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group-showed no eggshell, dense granular shape and adhesion together in string. In addition, no significant difference in the morphological structure of adult worms stained with Hematein-Eosin was observed.Conclusions:The egg production capacity of S. japonicum was reduced in the earlier stage after BMP expression was reduced in laboratory, and the egg morphology was negatively influenced to some extent, which provided clues that BMP was probably involved in the oviposition behavior of S. japnicum. Moreover, the long-term and compensatory mechanism involved in this process needs more in-depth investigation.