Kinetic Studiesof DGE From Plutella Xylostella(L.) Immune Reponses Against Bacillus Thuringiensis And The Cloning Of PGRP-LB

Peptidoglycan recognition protein(PGRPs) are a kind of important pattern recognition receptors in insects immune system, which playing an important role in pathogenic microorganisms and mediating the signal pathway.To study the role of PGRPs genes inimmune pathogenic microorganismsin diamondback moth(Plutella xylostella). Inthe current research,after the virulence of Bt was determined, samples were collected at 12 h,18 h, 24 h and 36 h respectively after 1×108Bt infected P. xylostella. DGE(Digital Gene Expression Profile)data was measured by using high-throughput sequencing technologies. using bioinformatics analysis the DBM immune defense Bt infection related genes. Data analysis showed that there areremarkablely different during the post infection 12 h, 18 h, 24 h and 36 h, in the determination of Bt infect DBM after different time the number of(DGE), probes into the dynamics of Plutella xylostella immune defense Bt infect molecular mechanism;By rt-pcr RACE cloned PGRP LB1 and PGRP LB2 gene c DNA sequence, on the basis of the use of q RT PCR(quantative Real- time PCR) after studied the Bt infect PGRP LB1 and PGRP LB2 gene expression pattern of space and time;Use for feeding method of RNAi technology for PGRP LB1 and PGRP LB2 immune recognition of Bt infection has carried on the preliminary exploration.The main results were as follows:To explore dynamics mechanism of DBM immune system after Bt infection, this study measured the Bt infection based on RNA- Seq technology DGE after different time.In the genome as a background, compared the analysis of the diamondback moth immune defense Bt infection related genes and gene expression differences.DGE determination results showed that the Bt dealing with 6 h, 2573 differentially expressed genes, of which 747, up regulated and 1826 down regulated;Immune related genes, 249, of which 5 up regulated and 97 down regulated.Bt when handling 12 h, 1582 differentially expressed genes, of which 1190 up regulated and 392 down regulated;Immune related genes, 249, of which 8 up regulated and37 down regulated. Bt when handling 18 h, 1036 differentially expressed genes, of which 568 up regulated and 468 down regulated;Immune related genes, 249, of which 7 up regulated and 51 down regulated..Bt when handling 24 h, 3404 differentially expressed genes, of which 2746 up regulated and 658 down regulated;Immune related genes, 249, of which 30 up regulated and, 49 down regulated..Bt when handling 36 h, 4396 differentially expressed genes, of which 3877 up regulated and, 519 down regulated;Immune related genes, 249, of which 53 up regulated and 53 down regulated.By handling the differentially expressed genes from five points in a whole, PGRP family gene expression up regulated, Spatzle and Toll family expression level up regulated, Cactus expression down regulated clearly, Imd, relish expressionsignificantly reduced, In antibacterial peptide genes, only Lysozyme(Lys) family expression up regulated followed by the up regulation of peroxide enzyme and superoxide dismutase(sod).Results showed that the PGRP family in identifying Bt infection and the Toll signaling pathways, and combined with Lys family collaborative immune defense against Bt infection.Bt infection-induced Toll gene expression level and Imd gene expression showed that DBM immune defense against Bt infection is not only through the Toll but also by Imd, relish pathways to enhance immune defense ability.Secondly, nine PGRPs, according to the different treatment of RPKM value found significant differences between the two PGRP genes, namely PGRP LB1 and PGRP LB2, compared with PBS treatment 6 h, PGRP- LB1 at 6 h and 42 h each have a small peak at 69 and 35 times, PGRP- LB2 at 6 h and 42 h each have a small peak for 309 times and 309 times, showed that PGRP LB1 and PGRP LB2 in DBM immune recognition plays an important role in the process of Bt.To explore Px PGRP LB1 and Px PGRP LB2 gene expression patterns and sensitivity after induction of pathogenic microorganism, q RT- PCR method was used to detect the total RNA expression of Px PGRP LB1 and Px PGRP LB2 in DBM.The results showed that total RNA expression of Px PGRP LB1 and Px PGRP LB2 in DBM has a different degree of expression and PGRP- LB1 showedhigh expression level after 6 h of induction, then it reduced and after 18 h showed another other small peak; PGRP- LB2 showed high expression level after 18 h. The expression was also detected in five tissues of DBM, in the epidermis both genes showed high expressionlevel in 48 h, maximum, in the haemocytes also in the 48 h; in the midgut, for PGRP- LB1 genes in 18 h, achieved a peak while PGRP- LB2 in 48 h showed maximum peak.PGRP LB1 showed maximum expression level at 18 h in fat body while PGRP- LB2 in 6 h; in the malphigian tube, both genes showed high level of expression in 48 h.To explore the function of PGRP LB1 and PGRP LB2 in DBM immune recognition process against Bt and the primers were designed according to the PGRP LB1 and PGRP LB2 Unigene sequences. Px PGRP- LB1 gene length is 937 bp, the complete open reading frame of 594 bp encoding 197 amino acid residues, predicted mature peptide molecular weight 21.197 k Da and an isoelectric point of 5.48.Px PGRP- LB2 gene length is 772 bp, complete open reading frame of 612 bp, encoding 204 amino acid residues, predicted mature peptide molecular weight 22.114 k Da and an isoelectric point of 7.40. First 18 amino acids have the feature of signal peptide secreted protein, and presumed to be a secreted protein.Sequence alignment and the evolutionary relationship analysis showed that Px PGRP LB1 and Px PGRP LB2 belong to PGRP- LB family.In order to further explore Px PGRP LB1 and Px PGRP LB2 genesfunction in DBM immune system against pathogenic microorganisms, expression plasmids were successfully constructed expressing GFP, PGRP LB1 and PGRP LB2 ds RNA L4440- GFP, L4440-PGRP LB1 and L4440 PGRP- LB2, and successfully transferred into the strain HT115, 1 x 107 cfu/ml expression strains feeding Plutella xylostella, 24 h after feeding 1 x 105 cfu/ml of Bt bacteria, and the results indicate gene silence does have some effect. In summary, Px PGRP LB1 and Px PGRP LB2 are inducible proteins with a recognition effect on bacteria. Px PGRP LB2 has a stronger recognition effect than Px PGRP- LB1 and plays an important role in the immune system of DBM. This study not only complements the PGRP family members, but also discusses the role of DBM Px PGRP LB1 and Px PGRP LB2 genes in resistance to the pathogenic microorganisms.More importantly, it is the first use of RNAi feeding method applied against important agricultural pests like diamondback moth and lays a solid foundation in promotion of this method.