Cloning,localization And Immune Function Of MMPs Gene In Dugesia Japonica

Matrix Metalloproteinases(MMPs),a member of the zinc-dependent metalloproteinases protein family,are widely found in plants,animals and microorganisms.MMPs are involved in regulating numerous physiological processes,pathological processes,and immune responses by degrading extracellular matrix(ECM)and basement membrane(BM).Compared with invertebrate MMPs,vertebrate MMPs have diverse subtypes and complex functions.Therefore,it is difficult to study the function of MMPs in vertebrates.However,it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates.Dugesia japonica is the most representative species in the phylum flatophyta.It has been widely concerned for its strong regeneration ability and highly effective immunity.Studies have shown that MMPs are involved in the regeneration and homeostasis regulation of Schmidtea mediterranea.The biological function and regulatory mechanism of MMPs in the innate immune response of planarian are still unclear.In this study,the structural characteristics,evolutionary status and function of MMPs were preliminarily explored in Dugesia japonica.Cloning and evolutionary analysis of DjMMPs gene in Dugesia japonica: The fulllength DjMMPs of Dugesia japonica was cloned by RACE and RT-PCR technology.The ORF of DjMMP is 1746 bp and encodes 518 amino acids,which consists of a pro-peptide domain,a catalytic domain,a hinge domain,and a hemopexin domain.The ORF of DjMMP1 is 1488 bp and encodes 495 amino acids,which consists of signal peptide,a propeptide domain,a catalytic domain,a hinge domain,and a hemopexin domain.The ORF of DjMMP2 is 1503 bp and encodes 500 amino acids,which consists of signal peptide,consists of a pro-peptide domain,a catalytic domain,a hinge domain,and a hemopexin domain.In addition,the part of sequences of DjMMPB and DjMMP9 were cloned by the RT-PCR and RACE technology.Sequence analysis showed that DjMMPB and DjMMP9 have a typical MMPs structure.Compared with human MMPs,the "cysteine switch" and the conserved motifs of the prepeptide domains of DjMMPs were basically conserved by multiple sequence alignment.Multiple sequence alignment and phylogenetic analysis showed that these atypical DjMMPs were clustered with the MMPs homologous of Schmidtea mediterranea and DjMMP9 is more similar to vertebrates MMP9.These results indicated that DjMMPs are an atypical MMPs-like gene.Location of DjMMPs in Dugesia japonica: The in situ hybridization(ISH)was used to study the localization of DjMMPs in planarian.It is found that DjMMP1 and DjMMP2 were localized in the pharynx and a circle around the pharynx.The expression levels of DjMMP1 and DjMMP2 were up-regulated by q RT-PCR after LPS and LTA stimulation and injury at different time.It is found that DjMMP and DjMMPB were localized in the whole body parenchyma and the pharynx of planarian.DjMMP9 were localized in the whole body parenchyma,the pharynx and the brain of planarian.Compared with the normal planarian,the expression of DjMMP and DjMMPB weren't changed and the DjMMP9 expression decreased after X-ray irradiation.The expression of DjMMP,DjMMPB and DjMMP9 was decreased in H2B-defected planarian.These results suggested that DjMMP and DjMMPB have not only expressed in the whole body parenchyma and the pharynx of planarian,but also have a small amount of expression in neoblast cells.DjMMP9 have not only expressed in the whole body parenchyma,the pharynx and the brian of planarian,but also have a small amount of expression in neoblast cells.The expression of DjMMP,DjMMPB and DjMMP9 were up-regulated after X-ray irradiation by q RT-PCR.The expression of DjMMP,DjMMPB and DjMMP9 were up-regulated after temperature rise,starvation and injury at different time by q RT-PCR.This indicates that DjMMPs is sensitive to external stimuli.These results suggest that DjMMPs may be involved in planarian regeneration and innate immune response.DjMMPs participated in the innate immune response of planarian.RNAi was used to study the function of DjMMP1 and DjMMP2.The DjMMP1-defected and DjMMP2-defected planarians showed the unchanged phenotypic,but the DjMMP1-defected and DjMMP2-defected planarians showed the changed phenotypic and the delayed bacterial clearance after they were fed with bacteria.This suggests that DjMMP1 and DjMMP2 may be involved in the innate immune response of planarian.In order to futher study the function of DjMMP1,in vitro bacteriostasis experiments were performed with eukaryotic proteins.The results showed that DjMMP1 had antibacterial activity.These results suggest that DjMMPs may be involved in the innate immune response of planarian.DjMMPs participate in the regeneration of planarian.In order to further study the function of DjMMP by RNAi.The head of the DjMMP-defeceted planarian appears lesions,and even the head finally apoptosis,and finally lead to death.This suggests that DjMMP may be involved in the regeneration of planarian.In order to futher study the function of DjMMP in the regeneration of planarian,the study found that half of the heads of planarians were diseased after 3 days,and the heads were completely destroyed after 5 days.The DjMMP-defected causes delayed middle and tail regrowth and eye deformities.These results indicate that DjMMP is involved in planarian regeneration.In conclusion,we cloned for the first time the full length of three conserved MMPs family genes(DjMMP,DjMMP1 and DjMMP2)and the partial sequences of two conserved MMPs family genes(DjMMPB and DjMMP9)from Dugesia japonica.The genetic structure,evolutionary analysis and localization were studied.For the first time,DjMMP9 was found in planarian and its localization was carried out.It has been preliminarily proved that DjMMP1 and DjMMP2 could removed bacteria by lysozyme and participated in the immune response of planarian.It was found that DjMMP affected the regeneration ability and eye regeneration of planarian.This study not only enriches the related research progress of MMPs in invertebrates,but also provides data support and theoretical basis for further research on the regulation mechanism of MMPs.