Prokaryotic Expression Of The Immune-Related Genes And Its Relationship With Cry1Ac Toxin Resistance In Diamondback Moth,Plutella Xylostella (L.)

Diamondback moth(DBM),Plutella xylostella,as the first species of developing field resistance to Bacillus thuringiensis(Bt)Cry toxins,has a wide distribution and causes great economic losses to cruciferous vegetables and oil crop production.Previous studies on Bt resistance in DBM have often focused on receptor proteins in the gut.However,Bt preparations,whether they are toxic proteins or bacteria,are all foreign bodies after entering the body of DBM,which theoretically will cause an immune response.Therefore,whether the resistance of DBM to Bt is related to its immune system is a question worth exploring.In this study,some peptidoglycan recognition protein(PGRP)genes upstream of the DBM innate immune system signaling pathway and downstream antimicrobial peptide(AMP)genes were cloned.By feeding the proteins expressed by the above-mentioned immune-related genes,the possibility of relating to Bt resistance in DBM were studied.The results were as follows.1.A large number of base mutations were found existing in the PxPGRP1 gene both in Cry1Ac-sensitive strain(DBM1Ac-S)and Cry1Ac-resistant strain(DBM1Ac-R),and these mutations also caused changes in their coding amino acid sequences.There are 8 kinds of CDS in the DBM1Ac-S,and 56 base mutation sites between different sequences,and corresponding,7 kinds of amino acid sequences are encoded by them.Meanwhile there are8 amino acid mutation sites in the PGRP Domain.One of the peptide chains ends translation early due to base mutation,and does not have a complete PGRP Domain.There are 5 kinds of CDS in the DBM1Ac-R,35 base mutation sites between different sequences,and one kind of the CDS has a 6-base(TTACAG)insertion after the 382 nd base.Similarly,4 kinds of amino acid sequences are encoded by them,and 7 amino acid mutation sites in the PGRP Domain.One of the peptide chains has a 2 amino acid(VT)insertion in the PGRP Domain.Due to the large number of mutations in the PGRP Domain,the role of PxPGRP1 expressed in different sequences in the immune system of DBM may differ theoretically.2.The complete CDS(903bp)of PxPGRP3 gene was cloned for the first time,which encoding 300 amino acids,with a molecular weight of 33.5 k Da and an isoelectric point of7.38.There is no signal peptide in this peptide chain,but amino acids from 92 to 114 are transmembrane regions,amino acids from 1 to 91 are located inside the membrane,and amino acids from 115 to 300 are located outside the membrane.The amino acids from 150 to 278 have a PGRP Domain and also a N-acetylmuramoyl-L-alanine amidase conserved domain(Amidase-2).PxPGRP3 belongs to PGRP-LF family.3.The Px Defensin(Px Def)and Px Moricin(Px Mor)genes were cloned among the DBM1Ac-S,the DBM1Ac-R,and the DBM field strain(DBM-F).The complete CDS of Px Def is 360 bp,encoding 119 amino acids,a molecular weight of 13.5 k Da,and an isoelectric point of 4.45.The complete CDS of Px Mor is 198 bp,encoding 65 amino acids,a molecular weight of 6.9 k Da,and an isoelectric point of 12.01.Simultaneously,the Px Cecropin(Px Cec)gene was cloned from the DBM1Ac-S: complete CDS is 198 bp,encoding 65 amino acids,a molecular weight of 7.1 k Da,and an isoelectric point of 12.03.Although these genes have base mutations between different strains or within the strain,these mutations neither do not cause changes in the amino acid sequence nor only have 1 or2 amino acid changes in the signal peptide region of the corresponding peptide chain,which do not affect the function of the corresponding mature peptide.4.By predicting the spatial structure of PxPGRP1,PxPGRP2 and PxPGRP3,we found that the PGRP Domain parts of the above three genes have similar spatial structures and are similar to other PGRP proteins that have been identified.Also,we predicted the spatial structure of Px Def,Px Mor and Px Cec,which have typical insect Defensin,Moricin,and Cecropin characteristics,respectively.5.We successfully expressed PxPGRP2,PxPGRP3,Px Def,Px Mor and Px Cec using the prokaryotic expression system,and collected purified soluble target proteins of PxPGRP2,Px Def,Px Mor and Px Cec.After feeding the larvae on these expressed proteins and Cry1 Ac toxin,we found that: in the absence of Cry1 Ac toxin,the Px Def,Px Mor and Px Cec have significant protective effects on DBM(the mortality is lower than the control,and the pupation rate,pupal weight,emergence rate and adult longevity are higher than the control).Expressed Px Def,Px Mor and Px Cec had no significant effect on mortality,and PxPGRP2 significantly increased mortality,in the presence of Cry1 Ac toxin.In summary,the PxPGRP1 gene is rich in polymorphisms,which may be related to the extremely strong environmental adaptability of DBM;PxPGRP3 is a membrane protein and is not secreted to function extracellularly like many other PGRPs,simultaneously,it belongs to the PGRP-LF gene family and may have a down-regulating effect on the IMD immune signaling pathway;Px Def,Px Mor and Px Cec had no significant effect on Bt resistance of DBM,but they had significant protective effects against DBM when dealing with the complex microbial environment.The protein expressed by PxPGRP2 could significantly increase the insecticidal activity of Cry1 Ac toxin.This study provides new ideas and evidence to explain the Bt resistance mechanism of DBM,lays a theoretical foundation for the development of synergists targeting immune genes,and provides a theoretical basis for using the immune system to control pests.