Cloning And Expression Study Of Spsb From Dugesia Japonica

The SPSB family is comprised of four highly conserved proteins,each containing a C-terminal SOCS box motif and a central SPRY domain.Presently,Spsb genes have been found in mammals and in a few invertebrates,however,the specific functions of these genes are still unknown.In this study,we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb.The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals,and expression levels were also quantified in response to various stressors.The results are as follows:1.A cDNA of 1251 bp was identified by RACE PCR from both ends,which included a 5?-terminal untranslated region(UTR)of 105 bp and a 3?-terminal UTR of 87 bp.The 3? UTR was found to contain a canonical polyadenylation signal sequence(AATAAA)and a poly(A)tail.The open reading frame(ORF)was 1059 bp,which encoded a polypeptide of 352 amino acids.2.The ExPASy,TMHMM,SMART,Interpro and other online bioinformatics softwares were used to analyze the characteristics of the amino acid sequences from the Spsb gene.Results showed that: the deduced protein has a predicted molecular mass of 40.90 kDa and theoretical isoelectric point of 9.04.A sequence analysis of DjSPSB by SMART identified an SPRY domain and a SOCS-box.The predicted protein is hydrophilic with multiple phosphorylation sites and contains no signal peptide and transmembrane region.3.The phylogenetic trees were constructed based on amino acid sequence predicted from the gene by the Mr Bayes method.Phylogenetic analysis showed that DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1,Spsb2 and Spsb4.4.The spatial and temporal expression of the DjSpsb gene was analyzed by the whole-mount in situ hybridization,the sagittal cytosections and the RT-PCR.The results showed that: in sexually immature intact worms,DjSpsb transcripts were mainly located in the head and were more strongly found at the margin.Furthermore,DjSpsb transcripts were also detected in the guts;in sexually mature intacts,the hybridization signals were expressed in the testes and yolk glands besides the head.The results of the sagittal cytosections further confirmed that the strongest signals were expressed in the head and nervous tissues in the trunk.RT-PCR showed that DjSpsb m RNA was up-regulated at 1,3 and 5 days after amputation and reached a peak at 5 day.Afterwards,its expression levels reduced on day 7 post-amputation and were similar to the control.5.DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids.Above results suggest that the DjSpsb gene might be active in regeneration,especially,in central nervous system(CNS)formation and functional recovery during head regeneration,and it is also involved in the development of germ cells and stress responses in the planarians.