Identification Of Antheraea Pernyi Peroxiredoxin-1 And -2 And Their Regulatory Functions In Microbial Infection And DNA Damage

Back ground of study:Peroxiredoxins（Prxs）are a class of antioxidant proteins that detoxify peroxyni-trite,hydrogen peroxide,and organic hydroperoxides quickly and efficiently.Research involving human and animal cells reveal that Prxs can influence immune-related signal-ing as well as cellular oxidative stress responses.Cell proliferation,differentiation,em-bryonic development,immunological response,apoptosis,lipid metabolism,and cellular homeostasis are all influenced by members of the Peroxiredoxin family.Significance:In-depth research on insect innate immunity can help us better understand the syn-ergistic evolutionary relationship between pathogenicity and insect defensive systems,as well as pathogen infection patterns.Antheraea pernyi is a common wild silkworm that belongs to the order Lepidoptera.The goal of our research is to strengthen the immune system of A.pernyi so that it can effectively resist infectious pathogenic bacteria and reduce the loss to the silk industry.Furthermore,research on A.pernyi’s innate immune-related genes has substantial theoretical implications for enhancing disease resistance.In addition,various insects-related research is being conducted in order to provide a step-by-step reference for disease prevention and control.Methods:We used several techniques for the completion of this project.Polymerase chain reaction was used for the amplification of Peroxiredoxin 1（ApPrx1）and Peroxiredoxin 2（ApPrx2）from Chinese oak silkworm.Several bioinformatics techniques like BLAST,Expasy,Multiple Sequence Alignment MEGA 5.1 program for phylogenetic tree con-struction,Signal P 5.0 etc.were utilized for the analysis of sequence of ApPrx1 and Ap-Prx2.Quantitative real-time PCR（q RT-PCR）was utilized for the analysis of expression level of ApPrx1 and ApPrx2 in A.pernyi larvae in different tissues and after microbial stress.Recombinant proteins of ApPrx1 and ApPrx2 were prepared and purified using E.coli expression system.Recombinant ApPrx1 and ApPrx2 proteins were utilized for func-tional analysis like DNA cleavage assay by the metal-catalyzed oxidation system and recombinant proteins antioxidant activity etc.We also prepared double-stranded RNA of ApPrx1 and ApPrx2 later measured of H₂O₂ level,survival rate and antibacterial activity of in the ApPrx1 and ApPrx2 knockdown larvae compared with control.After exogenous H₂O₂ administration,we determined the m RNA expression profile of ApPrx1 and ApPrx2in hemocytes and the whole body of A.pernyi and compared it with the control group.Results:The cloning and characterization of two previously undescribed genes are pre-sented in this study.ApPrx1and ApPrx2 from Chinese oak silkworm（A.pernyi）were amplified using PCR.The ApPrx1 gene encodes a predicted 195 amino acid residue pro-tein.The calculated molecular weight of the ApPrx1was 21.8 k Da with an isoelectric point at 6.21.BLAST analysis revealed that ApPrx1 was highly homologous to that of Helicoverpa armigera peroxiredoxin 1（87.52%）and Trichoplusia ni peroxiredoxin 1（86.18%）,hence we named the identified protein ApPrx1.The ApPrx2 gene contains a687 bp open reading frame,encoding a predicted 288 amino acid residue protein.ApPrx2has an estimated molecular weight of 25.3 k Da and an isoelectric point of 7.70.showed the most similarity according to a blast analysis with Ostrinia furnacalis（76.20%）and Apis dorsata（71.69%）peroxiredoxin 2,hence it was named as ApPrx2.q RT-PCR anal-ysis revealed that the m RNA level of ApPrx1 was highest in the hemocyte,fat body and midgut.q RT-PCR analysis of different tissues of A.pernyi 5^th instar 3^rd day larvae showed that ApPrx2 expression was greatest in the fat body,followed by hemocyte and integu-ment.Furthermore,Beauveria bassiana（a species of fungus）,Escherichia coli（a species of gram negative bacteria）,and Micrococcus luteus（a species of gram positive bacteria）challenged fat bodies and hemocytes showed increased ApPrx1 transcript.The expression of ApPrx2 m RNA was dramatically induced following several microbial challenges（B.bassiana,E.coli,and M.luteus）.We expressed the recombinant ApPrx1 and ApPrx2proteins in E.coli,and then purified protein was used for functional studies.DNA cleav-age assay by the metal-catalyzed oxidation system using recombinant ApPrx1&ApPrx2showed that our proteins manifest the ability to protect supercoiled DNA damage from oxidative stress.To test the recombinant proteins antioxidant activity,the ability of Ap-Prx1 and ApPrx2 to remove H₂O₂ was assessed in vitro using recombinant ApPrx1 or ApPrx2 and DTT,while BSA+DDT served as a reference group.Our results revealed that both ApPrx1 and ApPrx2 help to remove H₂O₂ in vitro.q RT-PCR analysis revealed that the m RNA expression profile of ApPrx1 and ApPrx2 were decreased significantly decreased following double-stranded RNA treatment.Measurement of H₂O₂ was found significantly higher in the ApPrx1 and ApPrx2 knockdown larvae compared with control.We also found significantly less survival rate in the larvae in which ApPrx1 and ApPrx2were knockdown.While interestingly,in the ds RNA-administered larvae,the antibacte-rial activity was significantly higher compared with control.After exogenous H₂O₂ ad-ministration,we determined the m RNA expression profile of ApPrx1 and ApPrx2 in he-mocytes and the whole body of A.pernyi.The results revealed a significant increase in the expression profile of both ApPrx1 and ApPrx2 after 3 h and 6 h of injection.Conclusion:Altogether,our data suggest that both the ApPrx1 and ApPrx2 may have multiple functional roles in the physiology of A.pernyi since it protects supercoiled DNA damage from oxidative stress and controls antibacterial activity.