| Spodoptera frugiperda belongs to the Lepidoptera,Noctuidae and Spodoptera and is a major migratory pest worldwide.Highly resistant to both chemical and microbial pesticides,S.frugiperda has posed a major threat to crops such as corn,wheat,cotton and sorghum since it invaded China in 2018.Insects rely on their own strong immune system to resist the infection of pathogenic microorganisms,which weakens the control effect of microbial pesticides.Peptidoglycan-recognition proteins(PGRPs)are a class of important Pattern-recognition receptors(PRR),which are commonly found in insects and higher animals.It can specifically recognize and bind to Peptidoglycan(PG)on the surface of bacteria,regulate immune signaling,or exercise antibacterial function through amidase activity.In this theiss,the immune function of the PGRP-LB2 molecule of Fall armyworms was studied.The main findings are as follows:1.Sequence analysis of PGRP-LB2: The PGRP-LB2 gene(LOC118274949)is located on chromosome 11 of S.frugiperda,including 4 introns and 5 exons,with a 705 bp ORF,encoding 234 amino acids,and a signal peptide(aa: 1-16)and a conserved PGRP domain(aa: 47-186),with high parental relationship between amino acid sequences and PGRP homologous proteins of Lepidoptera.2.The tissue,development stage expression distribution and induced expression rule of PGRP-LB2 gene : RT-PCR detection showed that PGRP-LB2 was mainly expressed in 5 to6 years old S.frugiperda.In 5th instar larvae,it is mainly expressed in midgut.In blood cells,the expression of PGRP-LB2 increased 12 h after Escherichia coli injection.In the midgut,the expression of PGRP-LB2 was significantly increased 24 h after injection of E.coli,while the expression of PGRP-LB2 was significantly increased 6 h and 12 h after injection of Staphylococcus aureus.Increased expression of PGRP-LB2 was detected in blood cells,adipose bodies and midgut after injection of Beauveria bassiana spores at different times.3.Bacterial agglutination experiment: PGRP-LB2 recombinant protein can cause the agglutination of E.coli,S.aureus,Bacillus subtilis and Serrania marcescens,but the agglutination effect on bacteria is weakened after adding EDTA.Point mutant proteins Y101 A and H175 A have agglutination effect on E.coli and S.aureus,and Y101 A can also cause S.marcescens to agglutinate.4.Inhibition of bacterial growth experiment: PGRP-LB2 and H175 A proteins inhibited the growth of E.coli,S.aureus,and S.marcescens.Scanning electron microscopy showed that wild-type protein could cause damage to E.coli,S.aureus and S.marcescens.5.Analysis of amidase activity: The results of amidase activity analysis showed that PGRP-LB2 and point mutant proteins Y101 A and H175 A had degradation effects on E.coli peptidoglycan and S.aureus peptidoglycan.Wild-type PGRP-LB2 had the best degradation effect on E.coli peptidoglycan,and Y101 A still had significant degradation effect on E.coli peptidoglycan.6.The germination rate of conidia of B.bassiana showed that PGRP-LB2 decreased the germination rate of conidia of B.bassiana.In summary,the PGRP-LB2 gene can be significantly induced by bacteria and fungi,and the PGRP-LB2 protein has certain antibacterial activity,which can cause a variety of bacteria to agglutinate,has amide enzyme activity,and can inhibit the spore germination of B.bassiana.The results show that PGRP-LB2 is an important immune molecule in S.frugiperda. |
