Comparative Identification Of Immune Related Proteins At Different Development Stages Of Plutella Xylostella And Functional Study On Cactus Gene

Insects rely on their innate immune system to protect themselves against infectious pathogens.This immune response will change during insects developmental stages.Especially,it is more evident in insect metamorphosis,but the mutual interaction mechanism between the development and the immune system is unclear.In the present study,three developmental stages,including 4th instar larvae,Pupae and Adults,were selected and their protein libraries were constructed and compared by using isobaric tags for relative and absolute quantification(i TRAQ).The differently expressed immunity related proteins were identified in different developmental stages and the expression profiles were detected by q RT-PCR and Western blot.NF-?B signaling pathway played an important role in insect immunity and development,while cactus is an inhibitor of NF-?B.The full length c DNA sequence of Px Cactus was obtained by RT-PCR and RACE,and the expression profiles of Px Cactus was analysed by q RT-PCR and Western blot.The functional research of Px Cactus in regulating the expression of antimicrobial peptides was analysed by RNAi technology.The main results are as follows:1.Based on the protein differentiation at different developmental stages of P.xylostella detected by SDS-PAGE,the protein libraries during metamorphosis of P.xylostella were compared by using i TRAQ.4139 proteins were identified and 3254 proteins were quantitative informative.Differently expressed proteins were clarified based on a 95% confidence level,cutoff values of 2-fold.395 differentially expressed proteins were elucidated,including 203 up-regulated proteins and 192 down-regulated proteins between the Pupae and 4th instar larvae,512 differentially expressed proteins were identified,including 267 up-regulated proteins and 245 down-regulated proteins between the Pupae and Adult and 616 differentially expressed proteins were screened including 294 up-regulated proteins and 322 down-regulated proteins between the larvae and Adult from P.xylostella.GO enrichment analysis showed that different proteins associated with ATP synthesis,proton transport and redox processes.The results indicated that differential proteins mainly involved in energy metabolism.2.21 immunity related proteins were screened from these significantly differential proteins,including 5 pattern recognition proteins,3 antimicrobial peptides,6 serine protease inhibitors,1 PPO-like protein,1 catalase,2 peroxidases,2 SOD and 1 disintegrin.q RT-PCR and Western blot results indicated the our verification results are identical to the i TRAQ sequence data.3.The full length c DNA of Px Cactus(Gen Bank No.KY828920)was obtained by RT-PCR and RACE technology.The full-length sequence is 1868 bp,which consists of a 1053 bp open reading frame(ORF)encoding a 350 amino acids.The results of q RT-PCR indicated Px Cactus was expressed during the whole stages and tested tissues,with highest expression level in the 4th instar larvae and in the fatbody,respectively.q RT-PCR showed that Px Cactus was up-regulated significantly in fatbody after B.thuringiensis infection,and western blot analysis elucidated that Cactus protein in fat body of P.xylostella was increaed significantly after B.thuringiensis induction.4.qRT-PCR indicated that the transcription levels PxCactus was reduced after microinjection of ds RNA of Px Cactus from 8h to 48 h,and the downstream antimicrobial peptides genes,Cecropin1,Cecropin2,Cecropin3,Defensin,Gloverin,Lysozyme1 and Lysozyme2 were significantly up-regulated after 24 h post injection of ds Px Cactus.The results of RNAi showed that the expression of antimicrobial peptides was negatively regulated by the Cactus gene in P.xylostella.In conclusion,the immune-related proteins at different developmental stages of P.xylostella were screened by i TRAQ.q RT-PCR and Western blot results showed the relationship between immune-related proteins and developmental stages.RNAi result showed that the expression of antimicrobial peptides was negatively regulated by Cactus gene.This study not only provides a basis for elucidating the relationship between the immune system and development in P.xylostella,it also provides a theoretical basis for clarifying the function of Cactus in regulating of antimicrobial peptide expression in P.xylostella.