Characterization And Functional Analysis Of Pattern Recognition Receptor Associated Genes DjCTL And DjA2M In Planarian Dugesia Japonica

Planaria,as a typical representation of platyhelminthes,which have many transitional characteristics,is an important species for the study of the biological evolutionary history.In recent years,planarians have attracted the attenetion of biologists owing to their strong regeneration ability.These researches mainly focused on regeneration,nerve,toxicology and development,but with less on immune system.In this paper,thougth transcriptomic analysis for planarian Dugesia japonica using Hiseq 2000,c-type lectin(CTL)and alpha 2-Macroglobulin(A2M)were selected and performed future research of gene cloning,evolution and function analysis.Firstly,the transcriptome sequencing of planarian based on the second generation sequencing technology generated 42,877,438 raw reads.After quality control,removing impurity and assembling by Trinity,a total of 27,180 transcripts were obtained,which represent 21536 non-redundant All-unigenes.Among these,12119 unigenes accounted for 44.59% reached the length of 400 to 800 nt,and 51% sequences were proved ?80% of their sequence length can be matched with Uniprot protein databases,suggesting that the assembly quality was sufficiently high and the sequences we obtained previously reached the subsequent requirement for gene function analysis.Finally,combing with the KEGG and GO annotation results,246 immune related genes involved in 15 KEGG pathways associated immunity system were found in planarian,D.japonica.Secondly,a cDNA encoding DjCTL was cloned from planarian,D.japonica by RT-PCR and RACE technology.The gene spans 428 bp with a 5'untranslated region(UTR)of 48 bp,a 3'-UTR of 56 bp and an open reading frame(ORF)of 324 bp encoded a polypeptide of 108 amino acids.The deduced molecular mass of DjCTL is about 11.88 kDa with isoelectric point of 5.675.There is no signal peptide in the N-terminus of sequence whose C-terminus possesses a canonical polyadenylation signal sequence and a poly(A)tail.BLASTp reveals that,in this full-length sequence,only has a CRD,of which have four conservative cysteine residues composed two disulfide bond and two sugar binding sites: EPN and WND.Multiple sequence alignment found that CRD within DjCTL has high homology with other species,especially with Reg?? from human.In situ hybridization and slice immunohistochemistry demonstrated that the DjCTL mainly expressed in pharyngeal and body epidermis,speculated it may be involved in process of body's immune defenses.The subsequent upregulation of DjCTL in planarian induced by LPS and PGN future evidenced DjCTL involved in immune response.Hemagglutination and bacteria agglutination assay found that DjCTL can agglutinate rabbit red blood and combine with both gram negative and positive bacteria.Only depending on calcium can gram-negative bacteria agglutinate.However,the agglutination does not inhibit the growth of bacteria.Further study found that DjCTL can combine with LPS and PGN,which combining ability with LPS is greater than PGN.It also can be combined with mannose and galactose,which the combination ability of former is larger than the latter,as a result of the existence of EPN sequence.We speculate that,through above results,as a kind of PRRs in planaria,DjCTL may participate in the immune response.It is interesting that the results of real-time PCR with different regeneration time points and in situ hybridization show that,in early period of regeneration,DjCTL significantly high expression and mainly expressed in early blastema,suggesting that DjCTL play an important role in the early stages of regeneration,and may be connected with promoting mitosis.Finally,using RT-PCR and RACE technology,we have cloned a gene with total length of 5804 bp within a 5574 bp ORF,5'-UTR 128 bp,3'-UTR 102 bp,named DjA2 M.Its Nterminus contains a signal peptide of 20 amino acids,and C-terminus contains a canonical polyadenylation signal sequence and a poly(A)tail.Mature DjA2 M encod 1837 amino acids whose molecular weight is about 212 kDa with isoelectric point of 8.671.Thirty N-Linked glycosylation sites exist in this protein.Multiple sequence alignment found that,like other identified A2 M frorn different species,DjA2 M also contains three basic function regions: bait region,thioester region and receptor-binding region of which the corresponding conservative sites are also existed.The phylogenetic tree found DjA2 M located at the base of protostomia which conforming the important position of planarian in evolution.According to the results of in situ hybridization,DjA2 M only expressed in pharyngeal,suggesting that it may participate in planaria immune response.We discovered the expression level of DjA2 M raiseed obviously in planarias induced by LPS and PGN.After RNA interference to DjA2 M,planarian's fluid hemolysis activity is restrained along with body's fester,and positively related to the intensity of interference.Therefore,we hypothesized that DjA2 M participate in planaria immune response and may play an important role in the complement alternative pathway.