The Study On Insecticidal Activity Of N~6-(2-hydroxyethyl)Adenosine With Plutella Xylostella Adenosine Receptor Gene PxAdoR

Controlling pests with entomogenous fungi or its virulence factors is hot inpests biological control.The N6–(2–hydroxyethyl)–adenosine(HEA)had insecticidal activity to Plutella xylostella which was the metabolites of Paecilomyces cicadae(Mique)Samson.Based the specific fragment from differentially expressed of Plutella xylostella treated with HEA,the whole sequence of Plutella xylostella adenosine receptor(PxAdoR)gene was cloned by RT-PCR and RACE.Synthesis of dsRNA containing the purpose gene base on the PxAdoR gene.And with the fluorescent quantitative PCR to analyze relative quantitative expression of PxAdoR gene.Meanwhile,to make sure the PxAdoR protein function,the P.xylostella 2 instar larvaes were treated with HEA and the mammals of adenosine A2 a receptor antagonist SCH58261.The main results were as follows:1.Plutella xylostella adenosine receptor gene cloning and feature analysisBased the specific fragment from differentially expressed of Plutella xylostella treated with the metabolite of Ophiocordyceps sobolifera N6-(2-hydroxyethyl)-adenosine(HEA),the whole 1828 bp sequence of Plutella xylostella adenosine receptor(PxAdoR)gene was cloned by RT-PCR and RACE.The protein structure was predicted and analyzed using the bioinformatics software,it was found there were seven transmembrane helices which was belong to the G protein coupled receptors.The protein was hydrophobicity and instability with no signal peptide.Its molecular weight was 49.2 k Da.A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor(Dm Ado R)and a 32-35% identity with human AR.2.HEA affect the mortality and weight of plutella xylostella by activate PxAdoRThough second instar larvae showed a relatively higher expression of PxAdoR,we considered the second instar larvae for RNAi analysis.Quantitative RT-PCR analyzes,in the two independent RNAi experiments,showed that PxAdoR expression was reduced by 2.36-fold in comparison to GFP-fed control larvae 48 h after dsRNA feeding.The findings show that Pxds feeding resulted in a significantly inhibition in the expression of PxAdoR gene.After treatment with 1 mg/m L HEA,the relative PxAdoR m RNA level showed a highly significant,almost 4.3-fold(P < 0.001)increase compared to the control.This data shows that HEA affects PxAdoR gene expression and suggests that it could induce insecticidal activity via P.xylostella Ado Rs.HEA,at a dose of 1 mg/m L,was found to be lethal to the second-instar larvae of P.xylostella,and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA(PxAdoR knockdown,Pxds)or antagonist of AR(SCH58261)at 36,48,or 60 h.Especially at 48 h,the rate of growth inhibition of the PxAdoR knockdown group was3.5-fold less than that of the HEA group,and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group.