Effect Of Cry1Ac On PGRPs Expression In The Immune Receptor Gene Of The Diamondback Moth, Plutella Xylostella(L.)

The peptidoglycan recognition proteins(PGRP),as an important part of recognition receptor in insects,play an important role in the immune defense signaling pathway(Toll and IMD).In order to explore the effect of important agriculture pest DBM PGRP genes and phenoloxidase(PO),with periodically stressed by Cry1 Ac toxin,which produced by the highly pathogenic Bacillus thuringiensis(Bt).In our study,using Fujian Agricultural and Forestry University Diamondback moth database,the National Center for Biotechnology Information database and DBM transcriptome data,which provided by Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,and screening and correction of Plutella xylostella PGRPs sequences,cloning the Coding sequence of PGRP1,PGRP2 and PGRP3.Furthermore,the expression pattern of three PGRP genes with periodically stressed by Cry1 Ac toxin,was studied by Quantitative Real-time PCR(qRT-PCR).The main results were as follows:(1)After correcting the transcriptome,the PGRP gene of three strains(Cry1Ac-sensitive strains,Cry1Ac-resistant strains and Wuhan field strains)of P.xylostella was cloned and three PGRP the gene coding region were obtained.PGRP1,which gene length is 588 bp,encodes 195 amino acids residues.The predicted molecular weights in the Cry1Ac-sensitive strains,Cry1Ac-resistant strains,and Wuhan field strains were 21.49 KDa,21.42 KDa,and 21.50 KDa,respectively,and the isoelectric point was 8.29;PGRP2,complete open reading frame of 621 bp,encoding 206 amino acid residues,the predicted molecular weights in the Cry1Ac-sensitive strains,Cry1Ac-resistant strains,and Wuhan field strains are 22.36 KDa,22.36 KDa,and 22.50 KDa,respectively.The isoelectric points of the Cry1Ac-sensitive and resistant strains are both 7.59,and the isoelectric point of the field strains in Wuhan is 8.15;PGRP3,complete open reading frame of 792 bp,encoding 264 amino acid residues.The predicted molecular weight in the Bt-sensitive strains,Cry1Ac-resistant strains,and Wuhan field strains is both 29.51 KDa.The isoelectric point is 7.32.The predicted signal peptide of PGRP1 gene is from 1 to 21 amino acid,and PGRP2 gene is from 1 to 18 amino acid,while there is no predicted signal peptide of PGRP3.The predicted transmembrane region of PGRP3 is from 92 to 114 amino acid,while the predicted transmembrane region of PGRP1 and PGRP2 were not found.(2)The expression patterns of PGRP1,PGRP2 and PGRP3 in the midgut and fat body mixture of P.xylostella were investigated.The results of qRT-PCR showed that PGRP1,PGRP2,and PGRP3 have different expressions on the mRNA of the diamondback moth.in the midgut,1)the PGRP-1 mRNA levels of DBM1Ac-R strains and DBM field strains were reduced by ~80% and ~90% compared to DBM1Ac-S strains,respectively.2)the PGRP-2 mRNA levels of DBM1Ac-R strains were 3 times higher than DBM1Ac-S strains,while there was no significant difference in expression between the DBM-field strains and DBM1Ac-S strains.3)the PGRP3 mRNA levels of DBM1Ac-R strains and DBM field strains were respectively reduced by over 90% and ~70% compared to DBM1Ac-S strains.On the other hand,in the composition of fat body and epidermis,1)the PGRP-1 mRNA levels of DBM1Ac-R strains were increased 33 % compared to DBM1Ac-S strains,whereas DBM field strains were reduced ~90% compared to DBM1Ac-S strains.2)the PGRP2 mRNA levels of DBM field strains were over 17 times than DBM1Ac-S strains,whereas the PGRP2 mRNA levels of DBM1Ac-R strains showed an unapparent increase.3)the PGRP3 mRNA levels of DBM1Ac-R strains and DBM field strains were both reduced by over 90% compared to DBM1Ac-S strains.(3)The phenol oxidase activity assay results of three diamondback moth strains showed that the phenoloxidase activity of Cry1Ac-sensitive strains was 39.80 ng/ml,and the phenoloxidase activity of Cry1 Ac resistant strains was 50.58 ng/ml,the enzyme activity of the field strains in Wuhan was 45.83 ng/ml.In summary,in the immune defense system,phenol oxidase has a certain role,and different types of PGRP genes have different effects.under periodic Cry1 Ac toxin stress,PGRP1 gene showed obvious effects,while PGRP2 and PGRP3 needs to be further explored.The research results provide a theoretical basis for delaying the resistance of Lepidoptera pests to Bt preparations,and also lay a foundation for the study of new biological pesticides targeting the immune system of P.xylostella.