Cloning And Expression Of CDNA Encoding Odorant Binding Proteins In The Antennae Of Meadow Moth, Loxostege Sticticalis L.

Meadow Moth,Loxostege sticticalis L.(Lepidoptera:Pyralidae)is one of an eruptive agricultural and pastoral pest in North China.Recently,much progress had been gained on behavioral research of L.sticticalis,however,the molecular mechanism of the odor recognition in L. sticticalis was rarely reported.Study was carried on meadow moth's mechanism of preferential recognition for a suitable host plant during its ovipositing and feeding from the level of molecular biology.This study may offer some new ideas for the prevention or control the loss caused by meadow moth.It is known that odorant binding protein(OBP)in the antennae can play a role in perception of the information existing in environment,and these proteins are extensively distributed in olfactory receptors which belong to the antennae of insects.In this paper,the technologies of gene cloning and expression in E.coli were used for the study on OBE The major progress obtained from these studies are summarized as follows:(1)The general odorant binding protein 1(GOBP1)gene of L.sticticalis(the accession number is EU413989 in GenBank)was cloned by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplificaion of cDNA ends(RACE)techniques.Sequencing analysis shows that, LstiGBOP1 contains a complete coding sequece,with a signal peptide of secretory protein in eukaryote;The predicted protein is acid with 4 lipophilic regions;This protein has typical characteristics of odorant binding protein,and the whole sequence contains 6 conservative Cystine residues which forming 3 disulfide bonds;The deduced amino acid sequence shows a homology of 58.2%among lepidopteral insects.According to the known sequence,prokaryotic expression vector of LstiGOBP1 gene was constructed and successfully expressed in E.coli by inducing.The expressed products were consistent with the size of predicted recombinant protein by Western blot.(2)The general odorant binding protein 2(GOBP2)gene of L.sticticalis(the accession number is EU239360 in GenBank)was cloned by rapid amplificaion of cDNA ends(RACE)techniques.The nucleotide sequences of LstiGOBP2 have no difference in both sex.Sequencing analysis shows that, LstiGBOP2 contains a complete coding sequece,with a signal peptide of secretory protein in eukaryote;The predicted protein is acid with 4 lipophilic regions;This protein has typical characteristics of odorant binding protein,and the whole sequence contains 6 conservative Cystine residues which forming 2 disulfide bonds;The deduced amino acid sequence shows a homology of 81%among lepidopteral insects.According to the known sequence,prokaryotic expression vector of LstiGOBP2 gene was constructed and successfully expressed in E.coli by inducing,the expressed products were consistent with the size of predicted recombinant protein by Western blot.(3)The pheromone binding protein(PBP)gene of L.sticticaIis(the accession number is EU545482 in GenBank)was cloned by reverse transcription polymerase chain reaction(RT-PCR)and rapid amplificaion of cDNA ends(RACE)techniques.Sequencing analysis shows that,LstiPBP contains a complete coding sequece,with a signal peptide of secretory protein in eukaryote;The predicted protein is acid with 4 lipophilic regions;This protein has typical characteristics of odorant binding protein,and the whole sequence contains 6 conservative Cystine residues which forming 2 disulfide bonds;The deduced amino acid sequence shows a homology of 64%among lepidopteral insects. According to the known sequence,prokaryotic expression vector of LstiPBP gene was successfully constructed.(4)The pheromone binding protein FM1(PBPFM1)gene of L.sticticalis(the accession number is EU638330 in GenBank)was cloned by rapid amplificaion of cDNA ends(RACE)technique. Sequencing analysis shows that,LstiPBPFM1 contains a coding sequece of 138 amino acids,with a cleavage site between the 18th and the 19th amino acids;The predicted protein is acid,and it has typical characteristics of oclorant binding protein,and the whole sequence contains 6 conservative Cystine residues which forming 3 disulfide bonds;The deduced amino acid sequence shows a homology of 57.2%among lepidopteral insects.(5)The pheromone binding protein FM2(PBPFM2)gene of L.sticticalis(the accession number is EU638331 in GenBank)was cloned by rapid amplificaion of cDNA ends(RACE)technique. Sequencing analysis shows that,LstiPBPFM2 contains a coding sequece of 69 amino acids;The predicted protein is acid with 1 distinct lipophilic region;This protein has typical characteristics of odorant binding protein,and the whole sequence contains 3 conservative Cystine residues;The deduced amino acid sequence shows a homology of 51.2%among lepidopteral insects.(6)The ribosomal protein L24(Rp L24)gene of L.sticticalis(the accession number is EU339126 in GenBank)was cloned by rapid amplificaion of cDNA ends(RACE)technique.Sequencing analysis shows that,LstiRp L24 contains a complete coding sequece;The predicted protein is alkaline;The deduced amino acid sequence shows a homology of 94.4%among lepidopteral insects.