Characterization Analysis Of PAPs Gene And MiRNAs Targeting In Plutella Xylostella

Phenoloxidase is a key enzyme in melanization reaction,and its activation depends on the serine protease pathway of the cascade reaction.Serine proteases exist in the form of zymogens in the body.When a pathogen invades and is recognized by a signal,a series of serine proteases are activated by a cascade,eventually leading to the activation of prophenoloxidase-activating protesaes(PAPs),which then cleaves and activates prophenoloxidase(PPO).PPO produces active phenoloxidase(PO),which lead s to the formation of melanin and a melanization reaction,it is a universal defense mechanism in insects.Plutella xylostella is an important agricultural pest.In this experiment,three melanin-related genes PAPs(named PAP1,PAP3,and PAP3a)were analyzed and screened from the results of the PULL-DOWN experiment of P.xylostella plasma.This article uses molecular biology methods to study their function and mechanism in the melanization reaction,as well as the regulatory relationship with mi RN A.The following research results have been obtained:Sequence analysis showed that PAP1 and PAP3 a contained a single clip domain,while PAP3 contained two clip domain at the amino-terminus followed by a trypsin-like serine protease domain at the carboxyl-terminus.Multiple sequence alignment results show,PAP1,PAP3,PAP3 a encoding the amino acid sequence clustered with the PAP of other lepidopteran insects(Helicoverpa armigera,Manduca sexta,Bombyx mori,etc.),having a high similarity,homology relationships closer.The RT-qPCR technology was used to detect the expression patterns of PAP1,PAP3,PAP3 a in P.xylostella and the expression pattern after microbial induction.The result shows:(1)PAP1 is highly expressed in the epidermis and hemocyte,PAP3 and PAP3 a are highly expressed in hemocyte,and the expression levels of other tissues are low.(2)The expression of PAP1 was higher in the pre-pupae and fourth instar larva stages of P.xylostella;PAP3 was higher in the female pupae and fourth instar larvae of P.xylostella;PAP3a was highest in the male adult stage of P.xylostella,followed by the fourth instar larval stage;the expression of genes will fluctuate with the development of larvae.(3)After infection by Bacillus thuringiensis and Metarhizium anisopliae,PAP1,PAP3,and PAP3 a expression can be induced in the midgut and fatbody tissue of P.xylostella,indicating that PAPs participate in the immune response of P.xylostella,and play an important role in resist the invasion of pathogen.From mi RNA sequencing results of P.xylostella after induction by Bt and Ma,10 mi RN As corresponding to the target of PAP1,5 mi RNAs correspondence to the target of PAP3,4 mi RN As correspondence to the target of PAP3 a were predicted.In order to further determine its regulatory relationship,9 mi RNAs corresponding to the target PAP1 were finally selected for verification,and a si CHECK? Vectors of PAP1 was constructed.To explain the function of PAPs,in this experiment,a prokaryotic expression plasmid was successfully constructed,the fusion protein was purified,and polyclonal antibodies anti-PAP1 and anti-PAP3 were successfully prepared.For its detection,the titer of anti-PAP1 and anti-PAP3 is 1: 12800.In order to verify the structure and function of the protein,this study mutated the activation sites(NGDR,QGNR,GSDR)of PAP1,PAP3,and PAP3 a into the commercial protease Factor Xa to recognize and cleave IEGR.SDS-PAGE detection showed that the zymogen band of PAP1 Xa with a molecular weight of 57.3 KDa decreased and a band with a molecular weight of 29.4 KDa appeared,and PAP1,PAP3,and PAP3 a proteins that could be activated in vitro were obtained.In this paper,three melanin-related genes PAPs,were screened from the results of the PULL-DOWN experiment in P.xylostella plasma.The regulatory relationship between target genes and mi RNAs was clarified,and PAPs were involved in the immune response of P.xylostella.Illuminate PAPs play an important role in the serine protease cascade and the mechanism of melanination.It also utilized insect immune system provides a theoretical basis for biological control.