Cloning,expression profiling and binding characterization of the OBP2 gene in the diamondback moth,Plutella xylostella( Lepidoptera: Plutellidae)
Author(s): CHENG Xiao-Juan, CAI Li-Jun, ZHENG Li-Shuang, QIN Jiang-Mei, HUANG Yu- Ping, YOU Min-Sheng, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, College of Life Sciences, Fujian Agriculture and Forestry University
Pages: 365-376
Year: 2016 Issue: 4
Journal: Acta Entomologica Sinica
Keyword: Plutella xylostella; odorant binding proteins(OBPs); olfactory; gene expression; qRTPCR; fluorescence competitive binding assay;
Abstract: 【Aim 】 Odorant binding proteins( OBPs) play crucial roles,functioning as transporting vectors of odors,in host location and oviposition selection for many insects. Cloning and identification of OBP genes and clarifying their ligand-binding characteristics of the compounds may help address the molecular mechanisms in P. xylostella. 【Methods】OBP2 was cloned from P. xylostella based on PCR approach. Signal peptide and transmembrane region were predicted and the nucleotide sequence of P.xylostella OBP2( Pxyl OBP2) was aligned with that of the homologs from other insects using DNAMAN.A neighbor-joining tree was constructed using MEGA5. 0. Stage- and tissue-specific expressions of Pxyl OBP2 were profiled by using real-time quantitative PCR( qRT-PCR). Prokaryotic expression vector was constructed to express the recombinant protein,and the purified protein was detected by SDS-PAGE.The protein binding affinity of Pxyl OBP2 with 39 compounds was analyzed using fluorescence competitive binding assay. 【Results】Pxyl OBP2 was successfully cloned and sequenced. Its ORF is 546 bp in length( Gen Bank accession no. KT070562),encoding 182 amino acids,and the encoded protein has six cysteine conserved domains,with the predicted molecular mass of 22. 24 k Da and isoelectric point of5. 69. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The expression profiling of Pxyl OBP2 exhibited a higher expression level in virgin male adults than in female adults and mated male adults,and the expression levels of Pxyl OBP2 in legs of male and female adults were higher than those in other tissues. The binding affinity test of Pxyl OBP2 to three sex pheromones and 36 different plant volatiles showed that Pxyl OBP2 could bind with Z-11-16: Ald,with a dissociation constant of48. 951 μmol / L,and with 11 plant volatiles,exhibiting a stronger capability of binding linalool and 1-nonanol with the dissociation constants of 4. 733 and 6. 861 μmol / L,respectively. 【Conclusion】The nucleotide and amino acid sequences of Pxyl OBP2 were characterized. Based on qRT-PCR and competitive binding test results,we infer that Pxyl OBP2 may play important roles in adult courtship of the moth,and host plant volatiles play a synergism role.
Pages: 365-376
Year: 2016 Issue: 4
Journal: Acta Entomologica Sinica
Keyword: Plutella xylostella; odorant binding proteins(OBPs); olfactory; gene expression; qRTPCR; fluorescence competitive binding assay;
Abstract: 【Aim 】 Odorant binding proteins( OBPs) play crucial roles,functioning as transporting vectors of odors,in host location and oviposition selection for many insects. Cloning and identification of OBP genes and clarifying their ligand-binding characteristics of the compounds may help address the molecular mechanisms in P. xylostella. 【Methods】OBP2 was cloned from P. xylostella based on PCR approach. Signal peptide and transmembrane region were predicted and the nucleotide sequence of P.xylostella OBP2( Pxyl OBP2) was aligned with that of the homologs from other insects using DNAMAN.A neighbor-joining tree was constructed using MEGA5. 0. Stage- and tissue-specific expressions of Pxyl OBP2 were profiled by using real-time quantitative PCR( qRT-PCR). Prokaryotic expression vector was constructed to express the recombinant protein,and the purified protein was detected by SDS-PAGE.The protein binding affinity of Pxyl OBP2 with 39 compounds was analyzed using fluorescence competitive binding assay. 【Results】Pxyl OBP2 was successfully cloned and sequenced. Its ORF is 546 bp in length( Gen Bank accession no. KT070562),encoding 182 amino acids,and the encoded protein has six cysteine conserved domains,with the predicted molecular mass of 22. 24 k Da and isoelectric point of5. 69. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The expression profiling of Pxyl OBP2 exhibited a higher expression level in virgin male adults than in female adults and mated male adults,and the expression levels of Pxyl OBP2 in legs of male and female adults were higher than those in other tissues. The binding affinity test of Pxyl OBP2 to three sex pheromones and 36 different plant volatiles showed that Pxyl OBP2 could bind with Z-11-16: Ald,with a dissociation constant of48. 951 μmol / L,and with 11 plant volatiles,exhibiting a stronger capability of binding linalool and 1-nonanol with the dissociation constants of 4. 733 and 6. 861 μmol / L,respectively. 【Conclusion】The nucleotide and amino acid sequences of Pxyl OBP2 were characterized. Based on qRT-PCR and competitive binding test results,we infer that Pxyl OBP2 may play important roles in adult courtship of the moth,and host plant volatiles play a synergism role.
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