Study On The Regulation Of Antimicrobial Peptides By GNBP2 From Plutella Xylostella(L.)

Gram-negative binding protein,GNBPs are important pattern recognition protein?PRP?,playing important roles in recognizing conserved surface determinants of pathogens and triggering complex signaling pathways,such as Imd and Toll signal pathway in invertebrates.The present study was to characterize and identify expression profile of the GNBP2 in diamondback moth,Plutella xylostella?L.?,which designed as PxGNBP2.Meanwhile,the transcriptional regulation of antibacterial peptides?AMPs?had been investigated to verify the function of PxGNBP2.In the present study,reverse transcription polymerase chain reaction?RT-PCR?and rapid amplification of cDNA ends?RACE?was used to obtain the complete cDNA sequence encoding PxGNBP2.The full-length cDNA of the PxGNBP2 was 1673 bp which consists of a 1473 bp open reading frame?ORF?encoding a 491 amino acid,included a 19 amino acid signal peptide for secretion.The calculated molecular mass was 52 kDa with pI 6.73.Based on the phylogenetic tree and sequence alignment analysis showed that The PxGNBP2 amino acid sequence of diamondback moth is closely related to Pieris rapae,Danaus plexippus plexippus and Papilio xuthus.Real-time quantitative reverse transcription-PCR?qRT-PCR?analysis showed that PxGNBP2 gene was expressed constitutively in whole life stage,with the highest expression level in pupa stage,with a higher level in the 4^th instar larvae.Tissue distribution showed that PxGNBP2 was mainly expressed in midgut detected by qRT-PCR.Microorganism such as,Metarhizium anisopliae,Paecilomyces fumosoroseus,Staphylococcus aureus,Bacillus thuringiensis,Escherichia coli DH5a or Serratia marcescens rear into the 4^th instar larvae of P.xylostella.The mRNA level of PxGNBP2increased significantly up-regulated at different time points compared to the control,but the degree of infection was different after microbial challenge,especially with the highest expression level at 12 h after the induction of S.marcescens.But the transcript of PxGNBP2 were not increased up-regulated after fungi induction.Recombinant PxGNBP2 was produced as a fusion protein using pGEX4T-1expression plasmid that with GST tag.The fusion protein was easily purified to homogeneity using GST column.The results of SDS-PAGE and western blot showed the purified recombinant PxGNBP2 with a molecular mass of a about 52 kDa which can strongly cross-react with anti-PxGNBP2 serum prepared in the New Zealand rabbit.The anti-PxGNBP2 serum titer was confirmed by ELISA was of 1:256000.To investigate the function of PxGNBP2 in P.xylostella by RNAi,the healthy 4^thh instar larvae were injected PxGNBP2 dsRNA 60 h.qRT-PCR result showed that the transcript level of PxGNBP2 was significantly decreased after 6 h injection.We showed that the target gene PxGNBP2 transcript level was specifically knockdown after feeding P.xylostella larvae with bacteria HT115?DE3?expressed dsRNA from 6h to 18h.Further investigate whether the PxGNBP2 protein regulated the downstream AMP genes,the transcripts of Defensin?Moricin?Gloverin,Cecropin1?Cecropin 2 and Cecropin 3 were significantly down-regulated in Midugut of P.xylostella.Our results demonstrated that PxGNBP2 was an inducible,acute-phase protein and it can be induced by Gram-positvie and Gram-negative bacteria and it played a vital role in regulating the expression of AMP genes in P.xylostella.Therefore,the study of PxGNBP2may establish the theoretical foundation for elucidating the expression mechanism of antimicrobial peptides.