Expression Patterns And Functions Of Peroxidasin From The Rice Stem Borer (Chilo Suppressalis)

Peroxidasins represent the peroxidase-cyclooxygenase superfamily and are closely related to chordata peroxidases subfamily and peroxinectins subfamily.The peroxidase-cyclooxygenase superfamily was defined and shown to occur in all kingdoms of life..The multi-domain protein we describe here,Peroxidasins,has an enzyme portion that has pronounced homology with vertebrate peroxidases.They are multi-domain proteins containing a heme peroxidase domain,type C-like immunoglobulin domains,typical leucine-rich repeats and a von Willebrand factor C module.Drosophila Peroxidasin is expressed by blood cells(hemocytes)and some of their derivatives,such as plasmatocytes,and is deposited in basement membranes.Besides the peroxidase domain,Peroxidasins also contain domains characteristic of proteins of ECM and has recently been identified as an indispensable player in basement membrane(BM)synthesis.To date,there has been litter in vivo study of Peroxidasin function,Especially in Chilo suppressalis.1.The structural characteristics of CsPeroxidasinAnalyses of cDNA and its amino acid sequences,By PCR and RACE-PCR-based strategies,The isolated cDNA clone encoding CsPeroxidasin contained a 4080 bp open reading frame,which encoded a peptide of 1359 amino acid residues,The protein contained the hallmark of insect Peroxidasin,The hydrophobic signal peptide,ends at a predicted signal peptide cleavage site,a heme peroxidase domain,type C-like immunoglobulin domains,typical leucine-rich repeats,The majority of the putative proteins of Arthropod Peroxidasins seem to contain(Drosophila and human)a C-terminal von Willebrand factor C,But rice stem borer do not contain a C-terminal von Willebrand factor C.2.Tissue and Developmental expression of CsPeroxidasinTotal RNA was collected from the head,epidermis,fat body,midgut,and Malpighian tubules of Sixth-instar larvae The CsPeroxidasin transcripts were found in all tissues of the sixth instar larvae screened in this study,The highest CsPeroxidasin level was found in the head,significantly higher than other tissues CsPeroxidasin transcripts were also abundant in larval midgut,epidermis and malpighian tubes,but relatively low in the fat body and hemocyte,the lowest expression is in fat body;In order to determine CsPeroxidasin exhibit developmental expression patterns,we analyzed basal expression in the egg,first,second,third,fourth,fifth,sixth instar larvae,prepupae,pupae and adults of C.suppressalis.The results clearly showed that CsPeroxidasin mRNA levels fluctuated throughout the entire life cycle.The results demonstrated that the higher expression level of CsPeroxidasin is in egg,first,second,third instar larvae,At the beginning of fourth instar larvae,the expression of CsPeroxidasin level gradually decreasesd and rised to the high level at 104th and 112th hour,then declined slowly.In the Fifth instar larvae,about 48th hour CsPeroxidasin expression achieved to a highest level.in the early sixth instar larvae,the expression of CsPeroxidasin indicated low levels,Then the level was increased by the development of C.suppressalis.The CsPeroxidasin expression had a higher level in Pre-pupa and pupa stage.The results also profiled the CsPeroxidasin mRNA had a higher level in male adult than those of female.3?The effects of injecting dsRNAs of C.suppressalisThe injection of dsRNA was incubation for 30 min at 25 ? before used.24h after dsRNA injection,The phenotypic effects were observed as cannot be pupation in from prepupa normally.When injecting dsRNA as compared with the controls,We injected 39 prepupas and 24 prepupas died before entering into the pupa stage.Only 2 of 17 prepupas died by injected GFP dsRNA.With the exception of the final phase of 6th instar larvae injected with 2 ug dsRNA,there were no significant phenotypic effects differences between the treatments with injectde dsRNA and GFP dsRNA.4?Expression of CsPeroxidasin in Sf9 cell line and CAT activity of recombinant proteinsBy using Bac-to-Bac baculovirus expression system.The recombinant proteins were successfully expressed in Sf9 cell line and were detected by western blotting with anti-6×His-tag antibody.CAT activities in the enzyme preparations from blank Sf9 cells and Cs Peroxidasin were 65.54±9.35?15.83 ±3.49 U/mg protein,respectively.ANOVA analysis revealed that the CAT activities in the enzyme preparation from Cs Peroxidasin was significantly higher than those in blank cells.In summary,Our results suggest that CsPeroxidasin may plays an important physiological role in C.suppressalis.However,there are many questions regarding the importance and exact mechanism of Peroxidasin function,especially in insect.The preliminary results will provide a solid foundation for future research of insect Peroxidasins.