Molecular Mechanism Of Interactive Regulation On Immunity And Development By PxSerpin-7 In Plutella Xylostella

Serpin is a regulator of serine protease activity and is involved in many important life processes in insects,such as Toll signaling cascade and melanization in innate immune responses,indigestion,and metamorphosis.The study in our lab found that the immune response of Plutella xylostella.（Diamond back moth）is related to its development stage.The ability of insects to resist pathogens varies with developmental stages and is particularly evident during metamorphosis.Based on the genome of P.xylostella,and founded on the determination and assessment of transcriptome data and proteome data at different developmental stages of P.xylostella,We used bioinformatics to correlate transcriptome with proteome.The analysis revealed a Serpin（named Px Serpin-7）that is highly expressed in the transcriptome and proteome and regulated by micro RNA.It is speculated that Px Serpin-7 is involved in the regulation of P.xylostella development and immune defense.But Px Serpin-7 is mediated by the development of P.xylostella to regulate immune defense or regulating the immune response of P.xylostella to mediate development?To elucidate the molecular mechanism of Px Serpin-7 regulation of P.xylostella development and immune response,we used molecular biology and biochemical experiments to study its role in immunity and development.The results are as follows:Bioinformatics analysis of the amino acid sequence and structure of the Px Serpin-7which has the same expression pattern in the transcriptome and proteome of P.xylostella showed that the Px Serpin-7 is highly homologous to the Bm Serpin-7 and the domain is similar to Ms Serpin-7,indicates that the Px Serpin-7 belongs to the Serpin family and is presumed that the function is similar to the Ms Serpin-7.The spatiotemporal expression pattern and microbial induction expression of Px Serpin-7 in P.xylostella were studied by RT-q PCR.The results showed that:（1）Px Serpin-7 was highly expressed in larval hematocyte and with significant tissue specificity and developmental stage specificity.（2）The expression of Px Serpin-7 in the 3^rd instar larvae was significantly lower than that in other instar larvae and it fluctuates with the development of larvae,indicating that the Px Serpin-7 transcriptional expression pattern has an age-related developmental association.（3）Microbial induction experiments showed that bacterial and fungal pathogens infect P.xylostella could induce high expression of Px Serpin-7,indicating that Px Serpin-7 is inducible and involved in the immune response of P.xylostella.I constructed the prokaryotic expression plasmid and was successful to obtain recombinant protein and polyclonal antibody.The ELISA assay showed that the anti-Px Serpin-7 titer was 1:512,000.Western blot analysis showed that anti-Px Serpin-7 can effectively recognize Px Serpin-7 protein in the plasma of P.xylostella.To test whether the Px Serpin-7 gene is involved in the phenoloxidase cascade and the melanization,the recombinant protein Px Serpin-7 and the polyclonal antibody anti-Px Serpin-7 were incubated with plasma to determine PO activity in plasma.The results showed that:（1）Infection of P.xylostella larvae could induce the increase of PO activity in plasma of P.xylostella;the activity of PO in plasma of P.xylostella was activated by the cell wall components PGN-SA and the spores of Metarhizium anisopliae.rcp-Px Serpin-7 can not significantly effect to PO activity in plasma,but antibody anti-Px Serpin-7 significantly inhibited PO activity in plasma.（2）rcp-Px Serpin-7 can significantly induce the formation of black matter in plasma,while antibody anti-Px Serpin-7 significantly inhibits the formation of black matter in plasma.The results indicated that Px Serpin-7 is involved in the regulation of the melanization in the plasma of P.xylostella.To reveal the proteins interaction network of Px Serpin-7,and screen for proteins interacting with Px Serpin-7.This experiment expressed the recombinant Px Serpin-7 with His-tag and used PULL-DOWN technology to screen proteins directly or indirectly interacting with Px Serpin-7 and possible complex forms with Px Serpin-7.Mass spectrometry and bioinformatics analysis showed that Serpin1b,SP6,PAP1,PAP3,PAP3a,PPO1,PPO2,P450,PGRP-S2,Gloverin1,Lysozyme2,and other proteins,showed that these proteins interacted directly or indirectly with Px Serpin-7 and participated in the immune response of the P.xylostella.RNAi technology was used to study the function of Px Serpin-7 in P.xylostella.The ds RNA was injected into the P.xylostella larvae by microinjection,and the silencing effect of RNAi was examined by RT-q PCR and Western blot.The results showed that the expression rate of Px Serpin-7 decreased by 98%after injection of ds RNA 24 h.After injection of ds RNA,most of the P.xylostella could not be normalized,and the pupae could not be successfully emerged,indicating that the Px Serpin-7 is intricate in the metamorphosis of P.xylostella.This paper preliminarily clarified that Px Serpin-7 is involved in the immunity and development of P.xylostella.Px Serpin-7 plays an important role in melanization and the expression of antimicrobial peptides.The proteins interaction network of Px Serpin-7 was preliminarily constructed,which provided a basis for further analysis of immune and development related networks.It also utilized insect immune system and the development system provides a theoretical basis for biological control.