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Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
Agricultural Sciences in China
August 2010
2010, 9(8): 1160-1166
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors XIU Wei-ming1, 2, ZHANG Yi-fan1, 3, YANG Dian-lin1, DONG Shuang-lin2 and LIU Yu-sheng3 Agro-Environmental Protection Institute, Ministry of Agriculture/Farmland Ecosystem Observation and Experiment Station of Genetically Modified Organisms in Wuqing, Chinese Academy of Agricultural Sciences, Tianjin 300191, P.R.China 2 Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry of Agriculture/Entomology Department, Plant Protection College, Nanjing Agricultural University, Nanjing 210095, P.R.China 3 Plant Protection College, Shandong Agricultural University, Tai’an 271018, P.R.China 1
Abstract The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor (orthologue to the Drosophila melanogaster DOR83b) from the antennae of Spodoptera exigua and S. litura by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences. The two full-length cDNA sequences from olfactory receptor (OR) of male S. exigua and S. litura were named as SexiOR2 and SlitOR2, respectively. SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp, respectively, and both with deduced amino acid sequences of 473 residues. The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths, implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes. Key words: Spodoptera exigua, Spodoptera litura, olfactory receptor gene, sequence analysis
INTRODUCTION The insect olfactory system has evolved the capacity to recognize and discriminate an inordinate number of chemically distinct odors that signal the presence of food sources, oviposition sites, predators, or mating partners. Deciphering the molecular mechanisms involved in insect olfaction has been a particular challenge for several decades, especially in disease vectors and crop pest species (Malpel et al. 2008). The detection and transduction of olfactory signals requires olfactory receptors (ORs) located in the dendritic membrane of sensory neurons (Clyne et al. 1999; Vosshall et al. 1999; Zwiebel and Takken 2004). These ORs belong to the superfam-
ily of G protein-coupled receptors, which are characterized by seven membrane-spanning domains. Insect ORs are highly divergent in their sequences among and within species. As an exception, one particular OR is remarkably conserved among insect species (Hill et al. 2002; Krieger et al. 2003; Pitts et al. 2004). This particular receptor is first obtained from analyses of the genome databases of Drosophila melanogaster and refered to as DOR83b, and its conservation allowed the isolation of its counterparts from other insect by homology-based method. This conserved ORs have been identified from Diptera, Lepidoptera, Hymenoptera, and Coleoptera species (Hill et al. 2002; Krieger et al. 2002, 2003; Melo et al. 2004; Pitts et al. 2004; Xia and Zwiebel 2006; Wang et al. 2008; Jordan et al. 2009). Several func-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. XIU Wei-ming, Associate Professor, Tel: +86-22-23003119, Fax: +86-22-23003120, E-mail: [email protected]; Correspondence DONG Shuang-lin, Professor, Tel: +86-25-84395245, Fax: +86-25-84395245, E-mail: [email protected] © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60203-0
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
tions for this gene have been speculated, including that (1) forms a heterodimer with the ligand-sensitive OR, and (2) helps in the trafficking of ORs to the correct site in the dendritic membranes (Keller and Vosshall 2003; Krieger et al. 2003; Vogt 2005). Here, we have reported the molecular isolation and identification of OR83b ortholog genes from antennae of Spodoptera exigua and S. litura, two severe pests in many crops and vegetables, providing the basis for elucidation of the olfactory mechanisms in insects and development of novel pest management strategies.
MATERIALS AND METHODS Collection of insect tissues S. exigua and S. litura were reared at 26°C (14:10 h, L:D) in the laboratory on an artificial diet as described by Huang et al. (2002) and separated at pupal stage on the basis of sex. The antennae were collected from 3d-old male and female moths. These were excised at the base, immediately transferred into Eppendorf tubes, and immersed in liquid nitrogen. The antennae were stored at -75°C until further use.
Isolation of total RNA and cloning of gene Total RNA was extracted by homogenizing antennae using TrizolTM reagent (GIBCO, USA) following the manufacturer’s instructions. The first-strand cDNA for reverse transcriptional PCR (RT-PCR) was synthesized by following the protocol of MMLV reverse transcriptase (Promega, USA) with 2 μL of total RNA as the template. Then, 2 μL of cDNA was used in further PCR analysis. The degenerate primers for amplification of S. exigua and S. litura putative olfactory receptor were designed by the alignment of ORs sequences from Aedes aegypti (GenBank AY582943), Anopheles gambiae (GenBank AY363725), D. melanogaster (GenBank NM 079511), Heliothis virescens (GenBank AJ487477), Antheraea pernyi (GenBank AJ555486), and Bombyx mori (GenBank AJ555487). SexiOR2 sense primer: 5´CARCAYYTNAARGSNATHATG-3´, SexiOR2 antisense primer: 5´-TCNACCCARTAYTTDATNGC-3´. SlitOR2 sense primer: 5´-CAYTWYRYBTGYATGGG-3´, SlitOR2
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antisense primer: 5´-CCATHACYGAK GARCTCTC-3´. The reaction was performed with 50 μL reaction mixture containing 3 μL single-stranded cDNA, 2.0 mmol L-1 MgCl2, 0.2 mmol L-1 dNTP, 1.5 μmol L-1 of each primer, and 2.5 U Taq polymerase. The amplification of SexiOR2 cDNA segment was carried out under the following conditions: 94°C for 3 min, 6 cycles at 94°C for 50 s, 51°C for 1 min and 72°C for 40 s, with a decrease of the annealing temperature by 1°C per cycle. This was followed by 35 cycles at 94°C for 50 s, 45°C for 1 min, and 72°C for 40 s, and final incubation for 10 min at 72°C. The amplification conditions of SlitOR2 cDNA segment were 94°C for 3 min, 35 cycles at 94°C for 30 s, 51°C for 30 s and 72°C for 1 min, and final incubation for 10 min at 72°C. A rapid amplification of cDNA ends (RACE) procedure was employed to amplify the 5´ and 3´ end of the coding region using SMART RACE cDNA Amplification Kit (Clontech, Japan) following the instructions. Gene-specific primers (GSP) for 5´- and 3´-RACE were derived from the sequence of PCR products. The 5´and 3´-RACE GSP primers of SexiOR2 were 5´GGATTGTTGTTCTGCTGCCCAAAGTT-3´ and 5´CAGAAAAGATTCCTGATACGGTTGAC-3´. The 5´and 3´-RACE GSP primers of SlitOR2 were 5´TGTCATCCCGAAATCTTGCTGCG-3´ and 5´TTTCAATGCTATGAGCGGCACCAC-3´. The amplified PCR product was analysed with gel electrophoresis on 1.5% agarose gel. The sequences were identified by using the NCBIBLAST network server (http://blast.ncbi.nlm.nih.gov/ BLAST.cgi). The transmembrane regions were determined by using TMHMM software (http://www.cbs. dtu.dk/services/TMHMM).
RESULTS Identification of cDNA sequences encoding S. exigua and S. litura ORs The RT-PCR approach performed with the degenerate primers identified two cDNA fragments of 292 bp (SexiOR2) from S. exigua and 1 063 bp (SlitOR2) from S. litura. A RACE procedure was further employed to obtain full-length sequences of the two genes. The
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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sequences of SexiOR2 and SlitOR2 (Figs.1, 2) were deposited in GenBank with the accession no. of AY862142 and DQ845292, respectively. The isolated cDNA clone encoding SexiOR2 was 1 906 bp with an open reading frame for a polypeptide of 473 amino ac-
XIU Wei-ming et al.
ids and a molecular mass of 53.303 kD, while cDNA clone encoding SlitOR2 was 2 483 bp with 473 amino acids and a molecular mass of 53.267 kD. SexiOR2 and SlitOR2 were predicted to encode seven transmembrane helixs with the classical structure of ORs.
Fig. 1 cDNA sequence and predicted amino acid sequence of SexiOR2. The stop codon is indicated with an asterisk. The shaded areas show the site of transmembrane amino acids. The same as below.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
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Fig. 2 cDNA sequence and predicted amino acid sequence of SlitOR2.
Multiple sequence alignment The full amino acid sequences of SexiOR2 and SlitOR2 were aligned with other lepidopteran species (Fig.3). It
was found that SexiOR2 displays high homologies (98%) with SlitOR2 and SlittOR2. SlitOR2 is more identical with SlittOR2 (99%). SexiOR2 and SlitOR2 share 96% identity with MsepOR2, 95% with HassOR83b, HzeaOR83b
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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XIU Wei-ming et al.
Fig. 3 Alignment of deduced amino acid sequences of SexiOR2 and SlitOR2 with other species. -, missing amino; SexiOR2, SlitOR2, SlittOR2, MsepOR2, HassOR83b, HzeaOR83b, MbraOR2, HvirOR2, DindOR2, BmorOR2, PxylOR2, AperOR2, and MsexOR2 denote ORs from S. exigua, S. litura, S. littoralis, Mythimna separate, Helicoverpa assulta, H. zea, Mamestra brassicae, Heliothis virescens, Diaphania indica, Bombyx mori, Plutella xylostella, Antheraea pernyi, and Manduca sexta, respectively.
and HvirOR2, 92% with MbraOR2, and 79-91% with DindOR2, PxylOR2, BmorOR2, AperOR2, and MsexOR2.
DISCUSSION Since the first insect olfactory receptors gene was
identified in Drosophila melanogaster (DOR), the analyses of the more or less fully sequenced genome of D. melanogaster has identified a lot of candidate OR genes. These OR genes are highly divergent. In general, the DOR proteins show about 17-26% sequence identity, with only a few cases showing identity of 40-60%, presumably due to recent gene duplications, and shar-
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
ing striking low sequence identity with vertebrates and nematodes (Gao and Chess 1999; Smith 1999; Vosshall 2003). In the OR family of fruit fly, the protein called OR83b is clearly distinct from other ORs. Among the 62 Drosophila OR proteins, 61 of which are individually expressed in small nonoverlapping sub-populations of olfactory sensory neurons (OSNs) of the antenna and maxillary palps. OR83b is coexpressed with the conventional ORs in nearly all olfactory neurons (Dobritsa et al. 2003; Elmore et al. 2003; Krieger et al. 2003; Pitts et al. 2004). OR83b and its orthologue chemoreceptors are the highly conserved OR proteins, with high homologies (50-99%). This conserved OR proteins have been identified from Diptera, Lepidoptera, Hymenoptera, and Coleoptera species. Studies showed that OR83b functions as a helper in odor transduction by heterodimerizing with conventional ORs in the endomembrane system in OSNs, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs (Larsson et al. 2004; Jones et al. 2005, 2007; Nakagawa et al. 2005; Neuhaus et al. 2005; Benton et al. 2006). Therefore, studies on these highly conserved odorant receptors may allow novel strategies of pest control. A potential strategy in pest control might be explored by interfering with the expression of OR83b orthologue chemoreceptor in a moth species. Moreover, as the OR83b orthologue chemoreceptors are highly conserved among different insect pests, this control technology would be used to insect pests of broadspectrum.
Acknowledgements This work was supported by funds from the National Natural Science Foundation of China (30800725 and 30770278) and the Central Public Research Institutes Basic Funds for Research and Development (Agro-Environmental Protection Institute, Ministry of Agriculture, China).
References Benton R, Sachse S, Michnick S W, Vosshall L B. 2006. A typical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biology, 4, 240-257. Clyne P J, Warr G G, Freeman M R, Lessing D, Kim J, Carlson J R. 1999. A novel family of divergent seven transmembrane proteins: Candidate odorant receptors in Drosophila. Neuron,
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22, 327-338. Dobritsa A A, van der Goes van Naters W, Warr C G, Steinbrecht R A, Carlson J R. 2003. Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron, 37, 827-841. Elmore T, Ignell R, Carlson J R, Smith D P. 2003. Targeted mutation of a Drosophila odor receptor defines receptor requirement in a novel class of sensillum. The Journal of Neuroscience, 23, 9906-9912. Gao Q, Chess A. 1999. Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics, 60, 31-39. Hill C A, Fox A N, Pitts R J, Kent L B, Tan P L, Chrystal M A, Ravchik A, Collins F H, Robertson H M, Zwiebel L J. 2002. G protein-coupled receptors in Anopheles gambiae. Science, 298, 176-178. Huang C X, Zhu L M, Ni J P, Cao X Y. 2002. A method of rearing the beet armyworm Spodoptera exigua. Entomological Knowledge, 39, 229-231. (in Chinese) Jones W D, Cayirlioglu P, Kadow I G, Vosshall L B. 2007. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature, 445, 86-90. Jones W D, Nguyen T A, Kloss B, Lee K J, Vosshall L B. 2005. Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Current Biology, 15, R119-R121. Jordan M D, Anderson A, Begum D, Carraher C, Authier A, Marshall S D G, Kiely A, Gatehouse L N, Greenwood D R, Christie D L, et al. 2009. Odorant receptors from the light brown apple moth (Epiphyas postvittana) recognize important volatile compounds produced by plants. Chemical Senses, 34, 383-394. Keller A, Vosshall L B. 2003. Decoding olfaction in Drosophila. Current Opinion in Neurobiology, 13, 103-110. Krieger J, Klink O, Mohl C, Raming K, Breer H. 2003. A candidate olfactory receptor subtype highly conserved across different insect orders. Journal of Comparative Physiology (A), 189, 519-526. Krieger J, Raming K, Dewer Y M E, Bette S, Conzelmann S, Breer H. 2002. A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens. European Journal of Neuroscience, 16, 619-628. Larsson M C, Domingos A I, Jones W D, Chiappe M E, Amrein H, Vosshall L B. 2004. Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron, 43, 703-714. Malpel S, Merlin C, François M C, Jacquin-Joly E. 2008. Molecular identification and characterization of two new Lepidoptera chemoreceptors belonging to the Drosophila melanogaster OR83b family. Insect Molecular Biology, 17, 587-596.
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Melo A C A, Rützler M, Pitts R J, Zwiebel L J. 2004. Identification
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of a chemosensory receptor from the yellow fever mosquito, Aedes aegypti, that is highly conserved and expressed in olfactory
pp. 753-804. Vosshall L B. 2003. Diversity and expression of odorant receptors
and gustatory organs. Chemical Senses, 29, 403-410. Nakagawa T, Sakurai T, Nishioka T, Touhara K. 2005. Insect
in Drosophila. In: Blomquist G J, Vogt R G, eds, Insect Pheromone Biochemistry and Molecular Biology. Elsevier
sex-pheromone signals mediated by specific combinations of olfactory receptors. Science Express, 307, 1638-1642.
Academic Press, London. pp. 567-591. Vosshall L B, Amrein H, Morozov P S, Rzhetsky A, Axel R.
Neuhaus E M, Gisselmann G, Zhang W, Dooley R, Störtkuhl K, Hatt H. 2005. Odorant receptor heterodimerization in the
1999. A spatial map of olfactory receptor expression in the Drosophila antenna. Cell, 96, 725-736.
olfactory system of Drosophila melanogaster. Nature Neuroscience, 8, 15-17.
Wang N N, Xiao J H, Lü B, You Z H, Huang D W. 2008. Interand intra-individual length-variant heterozygotes of intron 2
Pitts R J, Fox A N, Zwiebel L J. 2004. A highly conserved candidate chemoreceptor expressed in both olfactory and
in Or83b-like gene unveil evolutionary instability in Philotrypesis sp. (Hymenoptera: Chalcidoidea: Pteromalidae).
gustatory tissues in the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences of the USA,
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© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
August 2010
2010, 9(8): 1160-1166
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors XIU Wei-ming1, 2, ZHANG Yi-fan1, 3, YANG Dian-lin1, DONG Shuang-lin2 and LIU Yu-sheng3 Agro-Environmental Protection Institute, Ministry of Agriculture/Farmland Ecosystem Observation and Experiment Station of Genetically Modified Organisms in Wuqing, Chinese Academy of Agricultural Sciences, Tianjin 300191, P.R.China 2 Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry of Agriculture/Entomology Department, Plant Protection College, Nanjing Agricultural University, Nanjing 210095, P.R.China 3 Plant Protection College, Shandong Agricultural University, Tai’an 271018, P.R.China 1
Abstract The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor (orthologue to the Drosophila melanogaster DOR83b) from the antennae of Spodoptera exigua and S. litura by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences. The two full-length cDNA sequences from olfactory receptor (OR) of male S. exigua and S. litura were named as SexiOR2 and SlitOR2, respectively. SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp, respectively, and both with deduced amino acid sequences of 473 residues. The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths, implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes. Key words: Spodoptera exigua, Spodoptera litura, olfactory receptor gene, sequence analysis
INTRODUCTION The insect olfactory system has evolved the capacity to recognize and discriminate an inordinate number of chemically distinct odors that signal the presence of food sources, oviposition sites, predators, or mating partners. Deciphering the molecular mechanisms involved in insect olfaction has been a particular challenge for several decades, especially in disease vectors and crop pest species (Malpel et al. 2008). The detection and transduction of olfactory signals requires olfactory receptors (ORs) located in the dendritic membrane of sensory neurons (Clyne et al. 1999; Vosshall et al. 1999; Zwiebel and Takken 2004). These ORs belong to the superfam-
ily of G protein-coupled receptors, which are characterized by seven membrane-spanning domains. Insect ORs are highly divergent in their sequences among and within species. As an exception, one particular OR is remarkably conserved among insect species (Hill et al. 2002; Krieger et al. 2003; Pitts et al. 2004). This particular receptor is first obtained from analyses of the genome databases of Drosophila melanogaster and refered to as DOR83b, and its conservation allowed the isolation of its counterparts from other insect by homology-based method. This conserved ORs have been identified from Diptera, Lepidoptera, Hymenoptera, and Coleoptera species (Hill et al. 2002; Krieger et al. 2002, 2003; Melo et al. 2004; Pitts et al. 2004; Xia and Zwiebel 2006; Wang et al. 2008; Jordan et al. 2009). Several func-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. XIU Wei-ming, Associate Professor, Tel: +86-22-23003119, Fax: +86-22-23003120, E-mail: [email protected]; Correspondence DONG Shuang-lin, Professor, Tel: +86-25-84395245, Fax: +86-25-84395245, E-mail: [email protected] © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60203-0
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
tions for this gene have been speculated, including that (1) forms a heterodimer with the ligand-sensitive OR, and (2) helps in the trafficking of ORs to the correct site in the dendritic membranes (Keller and Vosshall 2003; Krieger et al. 2003; Vogt 2005). Here, we have reported the molecular isolation and identification of OR83b ortholog genes from antennae of Spodoptera exigua and S. litura, two severe pests in many crops and vegetables, providing the basis for elucidation of the olfactory mechanisms in insects and development of novel pest management strategies.
MATERIALS AND METHODS Collection of insect tissues S. exigua and S. litura were reared at 26°C (14:10 h, L:D) in the laboratory on an artificial diet as described by Huang et al. (2002) and separated at pupal stage on the basis of sex. The antennae were collected from 3d-old male and female moths. These were excised at the base, immediately transferred into Eppendorf tubes, and immersed in liquid nitrogen. The antennae were stored at -75°C until further use.
Isolation of total RNA and cloning of gene Total RNA was extracted by homogenizing antennae using TrizolTM reagent (GIBCO, USA) following the manufacturer’s instructions. The first-strand cDNA for reverse transcriptional PCR (RT-PCR) was synthesized by following the protocol of MMLV reverse transcriptase (Promega, USA) with 2 μL of total RNA as the template. Then, 2 μL of cDNA was used in further PCR analysis. The degenerate primers for amplification of S. exigua and S. litura putative olfactory receptor were designed by the alignment of ORs sequences from Aedes aegypti (GenBank AY582943), Anopheles gambiae (GenBank AY363725), D. melanogaster (GenBank NM 079511), Heliothis virescens (GenBank AJ487477), Antheraea pernyi (GenBank AJ555486), and Bombyx mori (GenBank AJ555487). SexiOR2 sense primer: 5´CARCAYYTNAARGSNATHATG-3´, SexiOR2 antisense primer: 5´-TCNACCCARTAYTTDATNGC-3´. SlitOR2 sense primer: 5´-CAYTWYRYBTGYATGGG-3´, SlitOR2
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antisense primer: 5´-CCATHACYGAK GARCTCTC-3´. The reaction was performed with 50 μL reaction mixture containing 3 μL single-stranded cDNA, 2.0 mmol L-1 MgCl2, 0.2 mmol L-1 dNTP, 1.5 μmol L-1 of each primer, and 2.5 U Taq polymerase. The amplification of SexiOR2 cDNA segment was carried out under the following conditions: 94°C for 3 min, 6 cycles at 94°C for 50 s, 51°C for 1 min and 72°C for 40 s, with a decrease of the annealing temperature by 1°C per cycle. This was followed by 35 cycles at 94°C for 50 s, 45°C for 1 min, and 72°C for 40 s, and final incubation for 10 min at 72°C. The amplification conditions of SlitOR2 cDNA segment were 94°C for 3 min, 35 cycles at 94°C for 30 s, 51°C for 30 s and 72°C for 1 min, and final incubation for 10 min at 72°C. A rapid amplification of cDNA ends (RACE) procedure was employed to amplify the 5´ and 3´ end of the coding region using SMART RACE cDNA Amplification Kit (Clontech, Japan) following the instructions. Gene-specific primers (GSP) for 5´- and 3´-RACE were derived from the sequence of PCR products. The 5´and 3´-RACE GSP primers of SexiOR2 were 5´GGATTGTTGTTCTGCTGCCCAAAGTT-3´ and 5´CAGAAAAGATTCCTGATACGGTTGAC-3´. The 5´and 3´-RACE GSP primers of SlitOR2 were 5´TGTCATCCCGAAATCTTGCTGCG-3´ and 5´TTTCAATGCTATGAGCGGCACCAC-3´. The amplified PCR product was analysed with gel electrophoresis on 1.5% agarose gel. The sequences were identified by using the NCBIBLAST network server (http://blast.ncbi.nlm.nih.gov/ BLAST.cgi). The transmembrane regions were determined by using TMHMM software (http://www.cbs. dtu.dk/services/TMHMM).
RESULTS Identification of cDNA sequences encoding S. exigua and S. litura ORs The RT-PCR approach performed with the degenerate primers identified two cDNA fragments of 292 bp (SexiOR2) from S. exigua and 1 063 bp (SlitOR2) from S. litura. A RACE procedure was further employed to obtain full-length sequences of the two genes. The
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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sequences of SexiOR2 and SlitOR2 (Figs.1, 2) were deposited in GenBank with the accession no. of AY862142 and DQ845292, respectively. The isolated cDNA clone encoding SexiOR2 was 1 906 bp with an open reading frame for a polypeptide of 473 amino ac-
XIU Wei-ming et al.
ids and a molecular mass of 53.303 kD, while cDNA clone encoding SlitOR2 was 2 483 bp with 473 amino acids and a molecular mass of 53.267 kD. SexiOR2 and SlitOR2 were predicted to encode seven transmembrane helixs with the classical structure of ORs.
Fig. 1 cDNA sequence and predicted amino acid sequence of SexiOR2. The stop codon is indicated with an asterisk. The shaded areas show the site of transmembrane amino acids. The same as below.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
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Fig. 2 cDNA sequence and predicted amino acid sequence of SlitOR2.
Multiple sequence alignment The full amino acid sequences of SexiOR2 and SlitOR2 were aligned with other lepidopteran species (Fig.3). It
was found that SexiOR2 displays high homologies (98%) with SlitOR2 and SlittOR2. SlitOR2 is more identical with SlittOR2 (99%). SexiOR2 and SlitOR2 share 96% identity with MsepOR2, 95% with HassOR83b, HzeaOR83b
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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XIU Wei-ming et al.
Fig. 3 Alignment of deduced amino acid sequences of SexiOR2 and SlitOR2 with other species. -, missing amino; SexiOR2, SlitOR2, SlittOR2, MsepOR2, HassOR83b, HzeaOR83b, MbraOR2, HvirOR2, DindOR2, BmorOR2, PxylOR2, AperOR2, and MsexOR2 denote ORs from S. exigua, S. litura, S. littoralis, Mythimna separate, Helicoverpa assulta, H. zea, Mamestra brassicae, Heliothis virescens, Diaphania indica, Bombyx mori, Plutella xylostella, Antheraea pernyi, and Manduca sexta, respectively.
and HvirOR2, 92% with MbraOR2, and 79-91% with DindOR2, PxylOR2, BmorOR2, AperOR2, and MsexOR2.
DISCUSSION Since the first insect olfactory receptors gene was
identified in Drosophila melanogaster (DOR), the analyses of the more or less fully sequenced genome of D. melanogaster has identified a lot of candidate OR genes. These OR genes are highly divergent. In general, the DOR proteins show about 17-26% sequence identity, with only a few cases showing identity of 40-60%, presumably due to recent gene duplications, and shar-
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors
ing striking low sequence identity with vertebrates and nematodes (Gao and Chess 1999; Smith 1999; Vosshall 2003). In the OR family of fruit fly, the protein called OR83b is clearly distinct from other ORs. Among the 62 Drosophila OR proteins, 61 of which are individually expressed in small nonoverlapping sub-populations of olfactory sensory neurons (OSNs) of the antenna and maxillary palps. OR83b is coexpressed with the conventional ORs in nearly all olfactory neurons (Dobritsa et al. 2003; Elmore et al. 2003; Krieger et al. 2003; Pitts et al. 2004). OR83b and its orthologue chemoreceptors are the highly conserved OR proteins, with high homologies (50-99%). This conserved OR proteins have been identified from Diptera, Lepidoptera, Hymenoptera, and Coleoptera species. Studies showed that OR83b functions as a helper in odor transduction by heterodimerizing with conventional ORs in the endomembrane system in OSNs, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs (Larsson et al. 2004; Jones et al. 2005, 2007; Nakagawa et al. 2005; Neuhaus et al. 2005; Benton et al. 2006). Therefore, studies on these highly conserved odorant receptors may allow novel strategies of pest control. A potential strategy in pest control might be explored by interfering with the expression of OR83b orthologue chemoreceptor in a moth species. Moreover, as the OR83b orthologue chemoreceptors are highly conserved among different insect pests, this control technology would be used to insect pests of broadspectrum.
Acknowledgements This work was supported by funds from the National Natural Science Foundation of China (30800725 and 30770278) and the Central Public Research Institutes Basic Funds for Research and Development (Agro-Environmental Protection Institute, Ministry of Agriculture, China).
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