Cloning And Recombinant Expression Of Scavenger Receptor PxSR And Its Function On Diamondback Moth Plutella Xylostell

Plutella xylostella is one of the most important agricultural pests.The larvae of this moth cause damage of cruciferous crops.Plutella xylostella has the characteristics of short life cycle,strong reproductive ability and easily developing insecticides resistance,which makes the difficulty for control of this insect pest.Therefore,it is particularly important to find effective biological control means to kill the pest.The scavenger receptor is an important component of the natural immune system of insects.The study on the scavenger receptor is helpful to understand the function of the innate immune system of Plutella xylostella,which is beneficial to find new targets for the biological control of the moth.In this study,four scavenger receptor genes were found from NCBI database based on bioinformatics analysis and cloned by RT-PCR,prokaryotic expression system was employed to express recombinant protein,and its polyclonal antibodies were prepared.Purified recombinant proteins were used to study its function in vitro.The main results were as follows:1.Based on sequence information in the NCBI database,the c DNA of four scavenger receptors（PxSR89,PxSR50,PxSR35,and PxSR24）were cloned by RT-PCR.The ORF length of PxSR89,PxSR50,PxSR35,and PxSR24 was 1494bp,1653 bp,1548 bp and1539 bp,respectively.PxSR is a transmembrane protein localized on the cell membrane with a hydrophobic CD36 conserved domain.Molecular evolutionary analysis revealed four genes in a large clade with Lepidoptera.2.The full-length ORF and extracellular region of PxSR89 were expressed by using various prokaryotic expression vectors.Recombinant engineered bacteria of E.coli BL21（DE3）-pET22b/pET28a/pET29a/pET24a/pET32a-PxSR89 and E.coli BW25113-PRB1K-PxSR89 ORF were constructed as well as six recombinant engineered bacteria expressing extracellular region:E.coli BL21（DE3）-pET22b/pET28a/pET29a-PxSR89,E.coli BW25113-PRB1K-PxSR89,and E.coli Origami 2（DE3）-pET28a/pET29a-PxSR89.Recombinant expression revealed that no soluble targeted proteins were produced using the above recombinant vectors.E.coli BL21（DE3）-pET22b-PxSR89 strain with the highest expression for full-length protein and E.coli Origami 2（DE3）-pET28a-PxSR89 strain with the highest expression for extracellular region were selected for expression of recombinant proteins.The recombinant proteins were purified,and polyclonal antibodies were prepared using the purified recombinant proteins as antigens.The antibody titer against full-length protein and extracellular region of PxSR89was as high as 1:128000 and 1:32000,respectively.PxSR89 was expressed in both pupal and adults verified by Western blot and Immunohistochemical method.3.The purified recombinant proteins were used to antibacterial test.The results showed that the extracellular region of PxSR89 inhibited the growth of Sarcina lutea.Conlutination tests showed that the extracellular region of PxSR89 had agglutination ability against Staphylococcus aureus,Bacillus subtilis,Sarcina lutea,Escherichia coli and Bacillus thuringiensis in the presence of Ca²⁺.Bacterial binding assay found that both full-length and extracellular region of PxSR89 had binding activity against the test bacteria;Molecular docking results showed that the forces between PxSR89 and sucrose and peptidoglycan molecules are hydrogen bonding and hydrophobic forces,Binding test with ligand showed that full-length and extracellular region of PxSR89 had high binding activity against LPS,peptidoglycan and mannose;The glucose inhibition experiment found that the binding ability of PxSR89 extracellular region to Escherichia coli was weakened after glucose treatment,and had the best inhibitory effect on Sarcina lutea.As one of the most important members of insect pattern recognition receptors,scavenger receptors play an important role in the moth protection against foreign pests.In this study,we preliminarily investigated the function of the scavenger receptor PxSR on Plutella xylostell in vitro and verified its expression as a pattern recognition receptor in Plutella xylostell and its ability to bind bacteria and carbohydrates.This work lays the foundation for further exploration of the function of the receptor in vivo.