Effect Of IDGF And βGBP On Humoral Immunity Of Plutella Xylostella Against Paecilomyces Cicadae

The diamondback moth,Plutella xylostella is a serious agricultural pest that harms cruciferous plants,causing huge losses to agriculture.At present,the diamondback moth has significant resistance to many insecticides,including Bacillus thuringiensis.Therefore,the development of biological control technology has become an inevitable trend to control P.xylostella.Unlike entomopathogenic bacteria and viral infections,entomopathogenic fungi actively invade the insect epidermis and eventually kill the insects.Understanding the insect innate immunity is a key to develop biological control methods by entomopathogenic fungi.Due to the lack of acquired immunity,insects rely on their own natural immunity to resist the invasion of various pathogens.Insects innate immunity,including cellular and humoral immunity,is the first line of defense against insect bacterial and fungal infections.If insects lose their innate immunity,their survival will be affected.The recognition proteins and antimicrobial peptides play a vital role in insect innate immunity.The elucidation of the mechanism of the immunity of P.xylostella to pathogens can provide a theoretical basis for new approaches to biologically control this pest.In this thesis,the innate immune response of P.xylostella against an entomopathogenic fungus Paecilomyces cicadae challenges was surveyed.Among the immunity-related genes potentially involved in innate immunity,two recognition molecules imaginal disc growth factors(PxIDGF)and β-glucan-binding protein(PxβGBP)genes were cloned and sequences were analyzed by bioinformatics.Expression profiles of them in different tissues and to different pathogen infections were performed using qPCR.We expressed PxlDGF and PxβGBP in a bacterial expression system and then purify them.The binding patterns of these two proteins to different microorganisms(bacteria and fungi)were assayed.The agglutination and PPO active activities of these two proteins were also assayed.The sequences of five antimicrobial peptides(lysozyme,gleverin,cecropin,moricin and defensin)of P,xylostella were analyzed.Expression profiles of them in different tissues and to different pathogen infections were performed using qPCR.The main work and achievements of this study are as follows:1.Molecular characteristics and functional identification of PxlDGF:The length of the open reading frames(ORF)of PxlDGF was 1299bp,encoding 432 amino acids(aa)with a GH18 domain.Sequence analysis found two amino acis for chitinase catalytic activity were substituted.PxIDGF was expressed at high levels in the fat body.When the P.xylostella is challenged by Escherichia coli K12 and P.cicadae,PxIDGF mRNA levels were up-regulated.PxIDGF can bind Gram-positive Staphylococcus aureus and Gram-negative E.coli cells.Recombinant PxIDGF protein agglutinated S.aureus and E.coli K12 cells in a zinc-dependent manner.PxIDGF enhances the prophenoloxidase activation in the hemolymph mixed with S.aureus,E.coli K12 and P.cicadae.2.Molecular characteristics and functional identification of PxPGBP:The length of PxβGBP ORF was 1422bp,which encodes 473 aa with a CBM39 domain and a GH16 domain.The active sites of bacterial glucanase(essential for b-1,3-glucanase activity)were replaced with other amino acid residues in PxβGBP.PxβGBP was mainly expressed in the fat body.The mRNA level of PxβGBP was induced by exogenous microbes,such as S.aureus,E.coli K12 and P.cicadae.PxβGBP can bind bacteria and fungi cells;Recombinant PxβGBP protein can agglutinate bacteria and fungi in a zinc-dependent manner.PxβGBP can enhance PPO activation in the presence of exogenous microorganisms(S.aureus,E.coli K12 and P.cicadae).3.The molecular characteristics of PxAMP:(1)The length of ORF of Pxgloverin was 519bp,encoding 172 aa.Pxgloverin is mainly expressed in fat body and hemocytes and Pxgloverin expression can be induced by P.cicadae,S.aureus,and E.coli K12.(2)Pxcecropin ORF had 201 bp encoding 66 aa.Pxcecropin mRNA levels were up-regulated by S.aureus and E.coli K12 infection.(3)The mature peptide of Pxmoricin had 42 aa.Pxmoricin is mainly expressed in fat body,and the expression level of Pxmoricin in the fourth instar is significantly higher than in other stages.The mRNA level of Pxmoricin by P.cicadae infection was higher than other treatments.(4)Pxlysozyme is mainly expressed in midgut.Pxlysozyme expression can be induced by P.cicadae and E.coli K12.(5)Pxdefensin is mainly expressed in fat body,and the expression level after P.cicadae infection is significantly higher than that in other treatments.