Functional Identification Of C-Type Lectin (PxIML) In Innate Immune Response Of Diamondback Moth,Plutella Xylostella(L.)

Insects are the most diverse and widely distributed animal population on the earth,which is closely related to their innate immunity system to cope with different habitats.The innate immune response of insects begins with the non-self-recognition of pathogen surface conservative pathogen associated molecular patterns（PAMPs）by pattern recognition receptors（PRRs）.Plutella xylostella（L.）is an important lepidoptera pest that endangers cruciferous crops worldwide.Like other insects,it has a complete innate immune system.C-type lectins（CTLs）is an important pattern recognition receptor in insect innate immune system,which can mediate humoral immunity and cellular immunity in insect innate immune system.Seven C-type lectin genes were identified in the Plutella xylostella genome by comparative genomics in the early stage of our research group,and one of them containing two tandem CTLD（C-type lectins domain）domain,named PxIML（Gen Bank accession number:MT232645）was cloned.In this study,the sequence and structural characteristics of PxIML were analyzed,the expression patterns of PxIML in different developmental stages,different tissues and response to the stimulation of pathogenic microorganisms were studied,and its immune recognition function was verified by protein expression in vitro.The results are as follows:1.The ORF sequence of PxIML gene is 969bp,which encodes 322amino acid residues,in which 1-23 amino acids are signal peptides and two CTLD domains lacated in 30-157 amino acids and 171-306 amino acids.The theoretical molecular weight and isoelectric point of mature PxIML are 34.34KDa and 5.05,respectively.Sequence analysis showed that mature PxIML forms at least two pairs of disulfide bonds in each CTLD domain,as well as conservative Glu-Pro-Asn motifs(EPN,Glu¹⁰¹-Pro¹⁰²-Asn¹⁰³)and Gln-Pro-Asp motifs(QPD,Gln²⁵¹-Pro²⁵²-Asp²⁵³)related to sugar recognition.In addition,PxIML also contains N-glycosylation site(Asn⁸⁴,Asn¹⁹⁴)and O-glycosylation site(Ser²⁵,Thr²²⁵).Phylogenetic analysis showed that the similarity between PxIML and immulectin-3（AIR95999.1）of Ostrinia furnacalis was the highest.Structural prediction showed that there were 4α-helixs and 15β-sheets in the secondary structure of PxIML.At the same time,PxIML may also bind to Ca²⁺,Alpha-D-mannose,Raffinose and Alpha-D-glucopyranosyl ligands.2.The spatio-temporal expression patterns of PxIML were detected by q RT-PCR,and it was found that PxIML was expressed in all ages of P.xylostella,and the expression of PxIML in 3^rd instar was significantly higher than that in other stages.In addition,PxIML was also expressed in different tissues of the 4^th instar larvae of P.xylostella,and the expression in fat body was significantly higher than that in hemolymph and midgut.After Bacillus thuringiensis（Bt8010）challenge for 18 h,the expression of PxIML in whole insect and fat body was significantly inhibited.After Serratia marcescens-IAE6 challenge for 18 h,the expression of PxIML in whole insect and fat body was significantly up-regulated,but there was no significant difference in midgut.3.Escherichia coli BL21（DE3）pG-Tf2 strain and pET-28b vector were used to express recombinant PxIML,and high purity recombinant PxIML（r PxIML）was obtained.The r PxIML has strong binding activity to E.coli BL21,Enterobacter sp.IAE5,Serratia marcescens-IAE6 and Staphylococcus aureus,but hardly binds to Bt8010 and Pichia pastoris.The binding of r PxIML to E.coli BL21 does not depend on Ca²⁺,but the binding to other bacteria depends on Ca²⁺.In addition,r PxIML can directly bind to lipopolysaccharide（LPS）,peptidoglycan（PGN）,D-Galactose,mannose,N-acety-D-（+）-glucosamine and N-acetylneuram-inicacid.In the presence of Ca²⁺,r PxIML had agglutination activity against E.coli BL21,Enterobacter sp.IAE5,Bt8010 and S.aureus.The agglutination activity against E.coli BL21 was weaker than that of the other three microorganisms,however,r PxIML had no agglutination activity against S.marcescens-IAE6 and P.pastoris.The gel beads coated with r PxIML can promote the encapsulation of haemocytes to some extent.In addition,r PxIML can bind directly to the surface of haemocytes.The addition of r PxIML to plasma significantly enhanced the activity of phenoloxidase（PO）.Based on the above results,Our study confirmed that P.xylostella PxIML can be used as a pattern recognition receptor to recognize and bind pathogenic microorganisms,and has a wide range of recognition and binding spectrum.PxIML can not only enhance the activity of PO in hemolymph and participate in the melanization reaction,but also bind directly to the surface of haemocytes and mediate the encapsulation of haemocytes to some extent.These results suggest that PxIML plays an important role in the innate immune system of P.xylostella,it can mediate the pathogen recognition and subsequently cellular and humoral immunity.Our study not only provides a basis for further study on the function of C-type lectin of P.xylostella,but also provides a new potential target for the biocontrol technology of P.xylostella.