| Phitella xylostella is a great hazard to cruciferous vegetables and has caused enormous economic losses worldwide.It is very difficult to control Plutella xylostella in the field because of its short life cycle,strong reproductive ability,strong adaptability to the environment,and strong drug resistance.As a widely used microbial insecticide,the continuous application of Bacillus thuringiensis has caused more and more pests to develop resistance.In this study,a high-toxicity Bt strains FJAT-12,which was isolated by Agricultural Bio-Resources Institute was used to explore the response mechanism of the innate immunity system of Plutella xylostella to Bacillus thuringiensis,especially the role of prophenoloxidase activating system.The main results were as follows.Transcriptome sequencing analysis of the expression levels of genes in P.xylostella infected by Bacillus thuringiensis,a total of 973 differentially expressed genes were detected,most of them were involved in the process of protein metabolism,proteolysis and protein transport.Quantitative Real-time PCR was performed for screening for genes related to insect immunity.The results showed that,up-regulated genes included the members of Toll pathway:peptidoglycan recognition proteins PGRPs,protease PSH,Toll receptor,protease pelle;antibacterial peptide,the products of immune signaling pathway:lysozyme,gloverin,cecropin;immune related factor hemolin,and the serine protease inhibitor Serpins which has a negative regulation of the prophenoloxidase activating system.The down-regulated genes mainly included trypsin and chymotrypsin which were serine proteases that could hydrolyze proteins in the midgut of the insect.The down regulation of both the trypsin and chymotrypsin would reduce the proteolysis of ?-endotoxin of Bt in the insect midgutChanges of phenoloxidase PO content in Plutella xylostella after feeding on Bacillus thuringiensis or 8-endotoxin Cry2Ab were detected by ELISA.The content of PO increased firstly and then decreased in both of the treatment groups,and the highest content was observed when feeding for 12 hours.The expression levels of PAP3,PPO1 and PPO2 in P.xylostella were detected by qPCR.The results showed that the expression of PAP3 increased while the expression of PPOl and PP02 decreased.To further understand the function of the phenoioxidase activation system of Plutella xylostella,the prophenoloxidase activating proteinase gene PAP3 of Plutella xylostella and two prophenoloxidase gene PPO1 and PPO2 were cloned and bioinformatics analysis was performed.The sequencing results showed that the PAP3 gene is 1260 bp in length and encodes 417 amino acids.The predicted protein has a molecular weight of 45.7 kDa and an isoelectric point of 5.95.It belongs to the f'amily of clip-domain serine proteases with a clip domain in the amino terminus and a catalytically active domain in the carboxy terminus.The PPO1 gene is 2049 bp in length and encodes 682 amino acids.The predicted protein has a molecular weight of 78.5 kDa and an isoelectric point of 6.33.And,the PPO1 gene has a total length of 2091 bp and encodes 696 amino acids.The predicted protein molecular weight is 79.8 kDa and the isoelectric point is 6.34.Both phenoioxidase genes contain two copper ion binding sites,and each copper ion binding region contains three conserved histidine(His)residues.We successfully constructed the prokaryotic expression vectors of three genes,which laid the foundation for the further study on the activation mechanism of phenoloxidase and phenoloxidase inhibitor.This study mainly analyzed the response of the innate immune system of Plutella xylostella to Gram-positive Bt by transcriptome sequencing and molecular cloning,which could provid reference for the further exploration of the innate immune system of Plutella xylostella and the development of new pest inhibitors. |
PDF Full Text Request