The Clone,Expression And Function Analysis Of Dj14-3-3? And Dj14-3-3? Genes In Planarian Dugesia Japonica

As a typical representation of platyhelminthes,planarias occupy an important position in the evolutionary process,which have been developed as a powerful system for the study of development,evolution,regeneration and immunity.14-3-3 proteins are a family of highly conserved acidic proteins that expressed in all eukaryotic cells.14-3-3 proteins,serving as an important signaling molecule,play a key role in controlling many biological processes.In this paper,we first isolated and characterized two members of the 14-3-3family,namely,Dj14-3-3 ? and Dj14-3-3 ? and detected their functions during regeneration and immunity.The full-length c DNA of Dj14-3-3 ? and ? were obtained by PCR and RACE.As shown that a 1305 bp nucleotide sequence of Dj14-3-3 ? was deposited in the Gen Bank database under the accession number KY070276.Its 5'-untranslated region(UTR)is 398 bp in length and 3'-UTR is 124 bp long.The longest open reading frame(ORF)codes for a deduced protein consisting 260 amino acids with a predicted molecular mass of approximately 29.69 k Da.The Dj14-3-3 ? sequence consists of 949 bp(Gen Bank accession number KY078796)and its ORF potentially codes for a protein of 254 amino acids(?29.68 k Da).Its 5'-untranslated region(UTR)is 78 bp in length and 3'-UTR is 286 bp long.The protein signatures analysis showed that Dj14-3-3 ? contained two common14-3-3 protein signatures.The consensus tree was obtained with phylogenetic analysis using the MAGE 6.0,which showed that Dj14-3-3 ? and Dj14-3-3 ? were located at the base of the evolutionary tree respectively and conformed to the evolution order.The result of whole mount in situ hybridization showed that the Dj14-3-3 ? and ?m RNA were abundantly located in the pharynx of adult intact and regenerative planarians.In the cross sections,the positive signals are mainly detected in the inner and outer epidermis of planarian pharynx.As pharynx is an immune organ of planarians,so we speculate that Dj14-3-3 ? and ? may participate in the immune responses.RNA interference(RNAi)was performed to characterize the function of Dj14-3-3 ?and ?.qRT-PCR analysis showed that the expression level of Dj14-3-3 ? was significantly increased in the Dj14-3-3 ?-RNAi planarians.Conversely,Dj14-3-3 ? was also increased in the Dj14-3-3 ?-RNAi planarians.The results suggest that Dj14-3-3 ? and ? have a mutual compensation in planarians.qRT-PCR was performed to analyze the transcription kinetics of Dj14-3-3 ? and ? in planarians challenged with pathogen-associated molecular patterns including LPS,PGN,?-Glu and Poly(I:C).The results demonstrate that Dj14-3-3 ? and ? can be induced to a high level by the LPS,PGN,?-Glu and Poly(I:C),so we speculate that they may participate in the early immune responses.During regeneration,the results of qRT-PCR show no obviously change of Dj14-3-3 ? and ? suggesting that Dj14-3-3 ? and ? may not participate in the pharynx regeneration.The prokaryotic expression vectors of p ET32a-Dj14-3-3 ?,p GEx-Dj14-3-3 ?,p ET32a-Dj14-3-3 ? and p GEx-Dj14-3-3 ? were constructed.The fusion proteins were expressed and purified.Western blot results suggest that the size of fusion proteins are consistent with the target proteins.In addition,the anti-rabbit-6his-Dj14-3-3 ? antibody was gained by immunizing rabbit.The expression of Dj14-3-3 ? and Dj14-3-3 ? proteins give the way for the further studying of Dj14-3-3 ? and Dj14-3-3 ? at the protein level in the planarian regeneration and immune process.