The Effect On Olfactory Of Diamondback Moth, Plutella Xylostella Of Polymines

Olfactory systems play crucial roles in insect mate-seeking,location of food and habitat, oviposition,survival and reproductive success.The polyamines spermidine(spd) and spermine(spin),as well as their precursor amine putrescine(put),are low-molecular weight nitrogenous compounds that are natural constituents of living cells.Polyamines have been shown to modulate insect behavior.Here,we study the olfactory related proteins of diamondback moth,Plutella xylostella and the effect of polyamines on the expression of those protein genes.The main results are as follows.1 Molecular characterization and expression pattern of olfactory related protein genes from diamondback moth,Plutella xylostellaWe describe the identification and characterization of new cDNAs encoding two general odorant binding proteins(PxylGOBP1,GenBank:ABY71034 and PxylGOBP2,GenBank: ABY71035) from the antenna of diamondback moth,P.xylostella,by RT-PCR and RACE, The deduced amino acid sequences of PxylGOBP1 and PxylGOBP2 revealed mature proteins of 168 and 163 amino acids,respectively,with six cysteine residues in conserved positions relative to other known GOBPs.The alignment of the mature PxylGOBPs with other Lepidoptera GOBPs showed high sequence identity(70-80%) with other full-length sequences from GenBank.Sequence identity between PxylGOBP1and PxylGOBP2 was only 50%,suggesting that the two proteins belong to different classes of GOBPs already described from the Lepidoptera;A new cDNA encoding a pheromone binding protein1 (PxylPBP1,GenBank:FJ201994),The deduced amino acid sequences of PxylPBP1 revealed a mature protein of 164 amino acids,with six cysteine residues in conserved positions relative to other known PBPs.The alignment of the mature PxylPBP1 with other Lepidoptera PBPs showed high sequence identity(70-80%) with other full-length sequences from GenBank;and a chemosensory protein1(PxylCSP1,GenBank: FJ361903),The deduced amino acid sequences of PxylCSP1 revealed a mature protein of 134 amino acids,with four cysteine residues in conserved positions relative to other known CSPs.The alignment of the mature PxylCSP1 with other Lepidoptera CSPs showed high sequence identity(70-80%) with other full-length sequences from GenBank. 2 Tissue Specificity and expression pattern of olfactory related protein genes RT-PCR analysis revealed that PxylGOBP1,PxylGOBP2 and PxylPBP1 genes are only expressed in antennae,and didn't expressed in other tissues such as head,leg,abdomen and wing.But we found that PxylCSP1 could expressed not only in antennae but also in other tissues such as head,leg,abdomen and wing. Real-time PCR analysis revealed that the expression levels of PxylGOBP1,PxylGOBP2 PxylPBP1 and PxylCSP1 were changed according to different sex,age,tissues and mated or virgin.The expression levels of these genes in eggs,larvals and pupae were just about 0.01%of in adult moths.The expression levels of PxylGOBP1 in eggs,larvals and papue were about 3 fold of those of PxylGOBP2.But the expression levels of PxylGOBP1 in adult moths were just about 1/5 of those of PxylGOBP2,the expression levels of PxylGOBPs in first larval were highest of whole larval period,this maybe related to their fetch behavior;The expression levels of PxylPBP1 in egg and pupa periods were higher than those in larval period,the expression levels were decreased with ages and was lowest in third larval,and increased until pupaed,the expression levels of PxylPBP1 in male adults were about 3-30 fold of those in female adults,and in mated adults(males and females) were higher than in virgin male and female moths;the expression levels of PxylCSP1 in egg and pupa period were significantly lower than those in larval period,just about 1/10,and in fourth larval was highest in whole larval period,which maybe because of the large appetite of fourth larval,in adult period,the expression levels of 4-8h moths after eclosion were higher,after mated,the expression level was higher that virgin in female,but lower in males.For above,the expression levels of olfactory protein genes were related to sex,age,tissues and mated or virgin,these whether or not related to the plasticity of olfactory-guided behavior of moths remains unknow.3 The effect on the expression of olfactory related proteins gene of polyamines and DFMOThe expression of PxylGOBP1 of unmated diamondback moth were increased after treated with-putrescine,but decreased in mated diamondback moth;the expression of PxylGOBP2 were decreased after treated with putrescine,only increased in 0 and 4 h post emergence female diamondback moth;the expression of PxylPBP1 was increased in mated males and decreased in mated females after treated with putrescine;the expression of PxylCSP1 of male and female moths in different periods were decreased after treated with putrescine,only increased in 16 h post emergence moths.The expression of PxylGOBP1 of unmated diamondback moth were decreased after treated with spderimine,but increased in 4 h post emergence males and mated females; The expression of PxylGOBP2 of unmated diamondback moth were decreased after treated with spderimine,but increased in 0 and 4 h post emergence females;the expression PxylPBP1 and PxylCSP1 of male and female moths in different periods were decreased after treated with spderimine,only the expression of PxylCSP1 were increased in 4h post emergence males and mated females after treated with spderimine.The expression of PxylGOBP1,PxylGOBP2 and PxylCSP1 of male and female moths in different periods were decreased and controlled in very low levels;the expression of PxylPBP1 of males were increased after treated with spermine,but decreased in mated males and females;the expression of PxylPBP1 were decreased after treated with DFMO, but increased in 4 and 16 h post emergence females.4 The prokaryotic expression and pufication of olfactory related protein of diamondback moth,P.xvlostellaBased on cloned PxylGOBPs,PxylPBP1 and PxylCSP1 genes information,The PxylGOBP1,PxylGOBP2,PxylPBP1 and PxylCSP1 genes were constructed into bacterial expression vector pET-22b(+) and successfully over expressed in E.coli BL21(DE3),mostly present as inclusion bodies.The four recombinant proteins showed an obvious band at about 19kD,22kD,18kD and 14kD through sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis.The induced cells were disrupted by sonication on ice.The isolate inclusion bodies were solubilized in 8 mol/L urea.Extracts were purified with a DE52 affinity column.In addition,the purified proteins were refolded and used to immunize mouse,and the antibodies with higher immuo-effeicency were obtained and tested by enzyme-linked immunosorbent assay.5 Scanning Electron Microscopy Observation on Sensilla of diamondback moth,P. xylostellaAntennae and the sensilla of P.xylostella were observed by scanning electron microscopy.The results showed that antennae were threadlike,and there were altogether twelve types of sensilla on the antenna:sensilla coeloconica,sensilla squamiformia, sensilla styloconica,sensilla bent-tipped,sensilla titanic,sensilla basiconicum,terminal hair,sensilla campullacea,sensilla rod-like and sensilla furcatea.And sensilla coeloconica includes five subtypes,the types,number and distribution of P.xylostella on both sexes are similar.The shape and distribution of sensilla were discussed.According to the distribution of sensilla,combined with morphological characteristics have been described in the literature,analysis of the possible functions of sensilla.To reveal the behavioral responses of P.xylostella to identify mechanisms and to explore the use of diamondback moth pest control agents act to lay a theoretical basis.