Gene Cloning,Evolution,Expression And Function Of DjPLSCR Gene From Planarian Dugesia Japonica

Planarians,as a typical example of platyhelminth of invertebrate animal,have attracted attention because of their strong powers of regeneration.The previous researchers focused on the aspects of regeneration,stem cells,development and nerve.In recent years,scientists are tremendous interested in the simple and effective innate immune system,which planarian has a high ability to clear the pathogen and low infection rate.Phospholipid scramblase（PLSCR）gene family encodes a kind of Ca²⁺-dependent multiple-palmitoylated type II endofacial plasma membrane protein,which contributed to a variety of physiological pathways,including lipid metabolism,blood coagulation cascade,structure and function maintenance of mitochondrial.Recent studies have found that PLSCR is involved in the process of mammalian immune response,which has antibacterial and antiviral activities.Whether PLSCR exists and which function it performs for invertebrate were still not determined unambiguously.In this paper,PLSCR was first cloned from Dugesia japonica,and the gene characterization,evolution and function of DjPLSCR in D.japonica was studied.The DjPLSCR cDNA was cloned by RT-PCR（reverse transcription PCR）and RACE from D.japoncia.The sequence analysis revealed that it contains an opening reading frame consisting of 726 bp encoding a putative protein of 241 amino acids with a predicted molecular mass of～28.7 k Da.Sequence analysis using Meg Align identified that it is a member of scramblases,because DjPLSCR containes the four conserved motifs of this family,including a DNA binding motif,a nonclassical NLS,a conserved Ca²⁺-binding EF-hand-like motif and a transmembrane motif.Phylogenetic analysis using MAGE 4 revealed that DjPLSCR,Caenorhabditis elegans scrm1 and scrm4 were clustered into an independent sub-clade,which was at the base of the tree,suggesting that the phylogenetic position of DjPLSCR is consistent with the location of D.japonica.To study the function of DjPLSCR,in situ hybridization was carried out.The result showed that m RNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx in planarians.Planarians are cut into three parts of the head,middle（including pharynx）and tail.Consistent with the whole-mount in situ hybridization mentioned above,the transcription levels of DjPLSCR in the middle fragment contained pharynx were significant higher than that in the head and tail fragments.The results showed that DjPLSCR m RNA expressed obviously in the planarian pharynx which is an important organ of immune system.Importantly,the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns[LPS、PGN、ploy（I:C）andβ-Glu],interferon and bacteria（Escherichia coli and Bacillus subtilis）,suggesting that DjPLSCR is involved in the immune response.In order to further study its function,the baculovirus eukaryotic expression vector of v Ac-DjPLSCR was successfully constructed using bac-to-bac technology,and DjPLSCR protein was identified by western blot,the result show that the DjPLSCR is about 28.7 k Da.Also,we constructed expression vector of DjPLSCR with N-terminal fusion GFP or 3Flag and observed the cellular localization of DjPLSCR by laser scanning confocal.The results show DjPLSCR is evenly distributed in cytoplasm and nucleus in untreated cells,and DjPLSCR mainly located in cell membrane following virus-induced.This not only shows that the expression changed of DjPLSCR after virus induction,further provided experimental support for DjPLSCR participating in the immune process of planarian.Subsequently,in order to study the mechanism of DjPLSCR involved in immune function,the cloning,expression and function of STAT which is the PLSCR transcriptional regulator was studied.Also,the interaction relationship between STAT and DjPLSCR was explored.In situ hybridization showed that m RNAs of DjSTAT are predominantly expressed in adult pharynx and head.The transcription level of DjSTAT was significantly upregulated when planarians were stimulated with LPS and poly（I:C）.We speculated that the DjSTAT is involved in the immune response.After degradation of DjSTAT by RNAi,planarians appeared apoptosis,which demonstate that DjSTAT plays a role in inhibiting cell apoptosis.At the same time,DjPLSCR was also down regulated,suggesting that DjPLSCR is a downstream protein of DjSTAT.The results indicate that planarian may be involved in the immune response through signal pathway mediated by DjSTAT-DjPLSCR.In summary,the gene structure,function and its mechanism of DjPLSCR were studied,which not only contribute to the further study on the mechanism of DjPLSCR participation in the immune process,but also expand the research of the gene structure and function.