Cloning, Expression And Functional Analysis Of Olfactory Related Genes Of The Rice Borer

The rice striped stem borer,Chilo suppressalis(Walker)(Lepidoptera:Pyralidae) is one of the most injurious insect pests office in China.Olfaction plays a criticle role in the detection of odour,seeking host and mating.Based on the study of antennal sensory organs,it's necessary to study the molecular mechanism of olfactory recognition.To explore the molecular mechanism of insect olfaction is not only helpful for elucidating the essential reason,but also offer an theoretical evidences in developing saver,effective attractant or deterrent.Meanwile,insects as ideal model organisms,study on their olfactory can help to understand the development of human olfaction.In this dissertation,â… used reverse transcription(RT)-PCR and RACE method to clone the olfactory related genes and express them in prokaryotic and eukaryotic system and studied their related functions.The primary results are as follows:1.A full-length cDNA encoding a general odorant binding protein 2(GOBP2) was cloned from the antennal of the rice striped stem borer Chilo suppressalis (Lepidoptera:Pyralidae)by the combination of reverse transcription-PCR(RT-PCR) and RACE-PCR.The cDNA contains a 489 bp open reading frame that encodes a 162 amino acid protein,termed Chilo suppressalis GOBP2(CsupGOBP2)which is similar in amount of amino acids and protein sequences of GOBP2 in other species of Lepidoptera and share the typical features of odorant-binding proteins.The predicted molecular weight(MW)and pI was 18.192 kDa and 5.08,respectively.The first 21 amino acids formed the signal peptide.Bacterical expression vector of pET30a/CsupGOBP2 was constructed and successfully expressed in BL21.The recombinant protein was purified by affinity chromatography and gel filtration and prepared the polyclonal antibody.RT-PCR showed that CsupGOBP2 mRNA is highly expressed in the adult female and male antennae and,so is CsupGOBP2 protein as revealed by Western blot analysis.Binding assays showed that N-Phenyl-1-naphthylamine(1-NPN)was specifically interacted with GOBP2.Competitive binding experiments revealed that Laurinaldehyd and the pheromone cis-11-hexadecenal acted as efficient competitors, indicating the most binding ability of cis-11-hexadecenaland and Laurinaldehyd among 17 odorants.2.Using the Bac-to-Bac baculovirus expression system,the cDNA encoding gobp2 of Chilo suppressalis was expressed Tn cells.His and GOBP2 fused protein was proved using the Western blot analysis with His-tag monoclonal antibody and polyclonal antibody,respectively.3.The partial sequences of SinGOBP2 and CmedGOBP2 were cloned and analysied.The results showed that gobp2 gene reflected the phylogenetic relationship of lepodoptera insects in some degree and consistent with the taxonomy of morphology.4.A full-length cDNA encoding a general odorant binding protein 1(GOBP1) was cloned from the antennal of Chilo suppressalis by the combination of reverse transcription-PCR and RACE-PCR.The cDNA contains a 522bp open reading frame that encodes a 173 amino acid protein,termed Chilo suppressalis GOBP1 (CsupGOBP1)which share the typical features of odorant binding proteins.The predicted MW and pI was 20.039 kDa and 4.84,respectively.The first 22 amino acids was the signal peptide.Different length transcripts of 3'UTR of CsupGOBP1 were first isolated.Bacterical expression vector of pET30a/CsupGOBP1 was constructed and successfully expressed in BL21.The recombinant protein was purified by affinity chromatography and prepared the polyclonal antibody.Competitive binding experiments revealed that all tested odorants had the weak competitive ability.5.A full-length cDNA encoding a pheromone-binding protein 2(PBP2)was cloned from the antennal of Chilo suppressalis by the combination of RT-PCR and RACE-PCR.The cDNA contains a 498 bp open reading frame that encodes a 165 amino acid protein,termed CsupPBP2.The predicted MW and pI was 18.544 kDa and 4.71,respectively.The first 21 amino acids was the signal peptide.Structure of pbp2 of Lepidoptera insects showed that all the reported genes exhibit the same structure characterized by 3 exons-2 introns,only different in the gene length.Extron 1 and 2 had exactly the same size,but extron 3 had great variation.The two introns were located at the same position but differed in length greatly.Prokaryotic expression vector of pET30a/CsupPBP2 was constructed and successfully expressed in BL21.The recombinant protein was purified by affinity chromatography and prepared the polyclonal antibody.Using the intrinsic fluorescence of tryptophan of CsupPBP2 to monitor the fluorescence quench,the results indicated that Z11-16:Ald quenched the fluorescence obviously and Z9:16:Ald,Z13-18:Ald have little quench effect to the intrinsic fluorescence.6.OR receptor gene was cloned from C.suppressalis and S.inferens using the degenerate primers designed according to the reported insect olfactory receptor gene. And the results showed that the gene was expressed in the antenna of C.suppressalis and S.inferens and shared high identity among insects.Expressed in the prokaryotic expression was unsuccess.7.cDNA library of Chilo suppressalis antenna was constructed.Identification by SfiI enzyme digestion and PCR amplification of CsupGOBP1,CsupGOBP2 and CsupPBP2 from cDNA library showed that the restructed library in the experiment is excellent and could afford for screening the related functional genes.