Molecular Characterization, Quantitative Analysis And Bacterial Expression Of Pheromone Reception Related Proteins In Spodoptera Exigua Hübner And Spodoptera Litura (Fabricius)

In insects, the olfactory systems have been evolved highly sensitive, enabling detect and discriminate among a diverse array of volatile chemicals that stimulus insect behavioral responses such as mate-seeking, location of food and habitat, oviposition. Olfactory systems play crucial roles in insect survival and reproductive success. The olfactory-based control strategies have developed an important means for pest control. The beet armyworm, Spodoptera exigua Hübner and common cutworm S. litura (Fabricius) (Lepidoptera: Noetuidae), are severe pests of various agricultural crops. Their sex pheromone of female moths have been identified as complex blends, and successfully used in pest population monitoring. However, it is not satisfactory to its use in pest control except with means of mass-traping and mating-disruption. Therefore, it is very important to gain insight on the molecular mechanism of the perception of female sex pheromone by the male. With the insights on the molecular machemism, it will be facilitated for molecular designing of specific regulator to these key olfactory proteins, and for further development of more effective pest behaviorally interfereing techniques. Here, we report the molecular identification and sequence analysis of PBP and odorant receptor genes from S. exigua and S. litura, and further experession and purification of the PBP protein. The main results are as follows.1. Molecular characterization and sequence analysis of PBP from S. exiguaPCR performed with a pair of degenerate primers designed on the conserved amino acid regions of FWK(R)EG(E)Y and HE(D)LN(K)WA in PBPs of other insects, afforded two cDNA products of 275 and 281 bp, respectively. They were called SexigPBP1 and SexigPBP2, respectively. A Rapid Amplification of cDNA Ends (RACE) procedure was employed to obtain full-length sequences of PBP1 and PBP2 from S. exigua. The two PBP sequences of SexigPBP1 and SexigPBP2 were deposited in GenBank with the accession numbers of AY540316 and AY545636, respectively. The full-length sequences of SexigPBP1 and SexigPBP2 are 1,077 and 819 bp, respectively. SexigPBP1 cDNA contains a 492 bp open reading frame that encodes a 164 amino acid protein. SexigPBP2 cDNA contains a 510 bp open reading frame that encodes a 170 amino acid protein. The two PBPs from S. exigua share low similarity (44% identity), but are very similar to PBPs of the same group from other insect species, respectively. In order to ascertain the intron/exon structure of the genes, the two PBP genes were further cloned and sequenced from the genomie DNA preparation. Both SexigPBP1 and SexigPBP2 genes contain two introns, and present a similar intron/exon structure. The ends of the introns have a typical GT-AG structure. The two introns in SexigPBP1 are located between Glu45 and Leu46 (intronl, 87 bp) and inside the codon for Aspl05 (intron2, 142 bp). The introns in SexigPBP2 are located between Glu49 and Met50 (intronl, 295bp) and inside the eodon for Asp110 (intron2, 644 bp). To investigate the presence of PBPs in different tissues of S. exigua, reverse transeription-polymerase chain reaction (RT-PCR) experiments were performed using specific primers. SexigPBP1 and SexigPBP2 are only expressed in S. exigua antennae. Real-time PCR was performed to compare the transcript levels of SexigPBP1 and SexigPBP2 from antennae of male to female moths. The transcription levels of PBP genes from antennae were higher in male moths than in females. The amounts of SexigPBP1 and SexigPBP2 mRNA in female antennae were about 39% and 73%, respectively, relative to those in male antennae.2. Cloning and sequence analysis of PBP from S. lituraSimilarly as in S. exigua, with RT-PCR technique, two cDNA fragments (275 and 281 bp) were cloned. They were named SlitPBP1 and SlitPBP2, respectively. Their full lengths were obtained using RACE procedure. The isolated cDNA clone encoding SlitPBP1 (GenBank accession numbers DQ004497) was 1,085 bp long and contained a 492 bp open reading frame for a polypeptide of 164 amino acids. The initial 23 amino acids are predicted as a signal peptide followed by the native protein of 141 amino acids with a molecular mass of 16,285 Da, and an isoelectric point of 5.14. The isolated cDNA clone encoding SlitPBP2 (804 bp) (GenBank accession numbers DQl14219) comprised the complete PBP precursor consisting of a 27 amino acids long signal peptide followed by the native protein of 143 amino acids with a molecular mass of 15,998 Da and a isoelectric point of 4.92. Both SlitPBP1 and SlitPBP2 genes also contain two introns from the genomic DNA preparation. The two introns in SlitPBP1 are located between Glu45 and Leu46 (intronl, 76 bp) and inside the codon for Asp105 (intron2, 104 bp). The introns in SlitPBP2 are located between Glu49 and Met50 (intronl, 437 bp) and inside the codon for Asp110 (intron2, 1251 bp). The ends of the introns have a typical GT-AG structure. The two PBPs from S. litura share low similarity (45% identity), but are very similar to PBPs of the same group from other insect species, respectively. RT-PCR experiments show SlitPBP1 and SlitPBP2 are only expressed in S. litura antennae. The transcription levels of PBP genes from male and antennae were compared with Real-time PCR. The amounts of SlitPBP1 and SlitPBP2 mRNA in female antennae were about 2.1% and 7%, respectively, relative to those in male antennae. Phylogenetic analysis showed that noctuid PBPs were classified into three distinct groups (Group 1-3), and SexigPBP1, SlitPBP 1 and SexigPBP2, SlitPBP2 belong to two different groups (Group 2 and Group 3), respectively.3. Bacterial expression and purification of PBPs from S. exiguaBased on cloned SexigPBP information, SexigPBP1 and SexigPBP2 were constructed into bacterial expression vector pET-30a(+) fused to N-terminal His-tag sequence in frame and successfully overexpressed in E. coli BL21 (DE3), mostly present as inclusion bodies. The two recombinant proteins showed an obvious band at about 22kD through sodium dodecyl sulfate-polyacrylamide gel eleetrophoresis (SDS-PAGE) analysis. The induced cells were disrupted by sonication on ice. The isolate inclusion bodies were solubilized in 8 Murea. Extracts were purified with a nickel affinity column. Cleavage of the fusion protein, with enterokinase and further purification led to an expected molecular weight protein (about 16kD). In adition, the purified SexigPBP1 was refolded and used to immunize mouse, and the antibodies with higher immuo-effeiceney were obtained and tested by enzyme-linked immunosorbent assay.4. Cloning and sequence analysis of odorant receptor genes from S. exigua and S. iituraBy RT-PCR method with a pair of degenerate primers designed against an alignment of several known insect odorant receptor sequences. Two cDNA fragments of odorant receptor gene, SexiOR2 and SlitOR2, were cloned and sequenced from the antennae of male S. exigua and S. litura. Their full lengths were obtained using RACE procedure. The full-length sequence of SexiOR2 (Genbank accession number AY862142) was 1,906 bp, contains a 1,419 bp bp open reading frame that encodes a 473 amino acid protein with a molecular mass of 53,303 Da and an isoelectrie point of 8.60. The full-length sequence of SlitOR2 (GenBank accession numbers DQ845292) was 2,483 bp long and also contained a 1,419 bp open reading frame for a polypeptide of 473 amino acids with a molecular mass of 53,267 Da and an isoelectric point of 8.63. SexiOR2 and SlitOR2 included seven putative transmembrane domains, which is the typical characteristic of G protein-coupled receptors. The sequence analysis indicated that the deduced amino acid sequences of SexiOR2 and SlitOR2 shared high identity with olfactory receptors from other previously reported insects (50%) and the homology between SexiOR2 and SlitOR2 reached 98%. Phylogenetic analysis showed that Drosophila melanogaster Or83b and its Orthologs reported from other insects were classified into three distinct groups, and noctuid olfactory receptors formed a group.In this paper, we describe the identification and characterization of pheromone reception related proteins from the antennae of S. exigua and S. litura, and report about the comparison and evolution of these proteins of orthologous proteins. Based on cloned SexigPBP information, bacterial expression vectors of SexigPBP1 and SexigPBP2 were constructed and successfully expressed. The recombinant proteins were purified by affinity chromatography. Furthermore, the antiserum was raised with the purified SexigPBP1. These results could be used to explore the molecular mechanism of pheromone reception in S. exigua and S. litura, and helpful for providing theoretical evidences in designing and developing new effective attractant or deterrent.