Biochemistry and Molecular Biology 39 (2009) 90–95

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Insect Biochemistry and Molecular Biology

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Pheromone-gland-specific fatty-acyl reductase in the adzuki bean borer, scapulalis (: )

Binu Antony a, Takeshi Fujii a, Ken’ichi Moto b, Shogo Matsumoto b, Mai Fukuzawa a, Ryo Nakano a, Sadahiro Tatsuki a, Yukio Ishikawa a,* a Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan b Molecular Entomology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan article info abstract

Article history: The adzuki bean borer , Ostrinia scapulalis, uses a mixture of (E)-11- and (Z)-11-tetradecenyl Received 4 August 2008 acetates as a sex . At a step in the pheromone biosynthetic pathway, fatty-acyl precursors are Received in revised form converted to corresponding alcohols by an enzyme, fatty-acyl reductase (FAR). Here we report the 6 October 2008 cloning of FAR-like genes expressed in the pheromone gland of female O. scapulalis, and the character- Accepted 10 October 2008 ization of a single pheromone-gland-specific FAR (pgFAR) and its functional assay using an insect cell expression system. As many as thirteen FAR-like genes (FAR-I–FAR-XIII) were expressed in the phero- Keywords: mone gland of O. scapulalis; however, only one (FAR-XIII) was pheromone-gland-specific. The deduced Fatty-acyl reductase Pheromone gland amino acid sequence of FAR-XIII predicted a 462-aa protein with a conserved NAD(P)H-binding motif in Functional assay the N-terminal region, showing overall identity of 34% with the pgFAR of Bombyx mori. A functional assay Ostrinia using Sf9 cells transfected with an expression vector containing the open reading frame of the FAR-XIII gene has proven that FAR-XIII protein has the ability to convert a natural substrate, (Z)-11-tetradecenoic acid, to a corresponding alcohol, (Z)-11-tetradecenol. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction mammalian FAR (Cheng and Russel, 2004) soon followed, no report on pgFARs in other insect species has appeared until now. In the Lepidopteran sex are mostly blends of fatty acid- present study, we aimed to clone and characterize the pgFAR gene derived C10–C18 compounds with 1–3 double bonds and a terminal of the adzuki bean borer, Ostrinia scapulalis, which uses a mixture of oxygenated functional group (Bjostad et al., 1987). Pheromones are (E)-11- and (Z)-11-tetradecenyl acetates (E11- and Z11-14:OAc) as synthesized in a pheromone gland, which is usually a modified a sex pheromone (Huang et al., 1997, 2002; Takanashi et al., 2005). membrane between the 8th and 9th abdominal segments, from We here report the successful cloning and functional assay of common fatty acids like palmitic acid via chain shortening by pheromone-gland-specific FAR from O. scapulalis. b-oxidation enzymes, introduction of a double bond by fatty-acyl desaturase, reduction by fatty-acyl reductase (FAR), and acetylation by acetyltransferase in the case of acetate pheromone or oxidation 2. Materials and methods by alcohol oxidase in the case of aldehyde pheromone (Tillman et al., 1999; Jurenka, 2003, 2004; Matsumoto et al., 2007). In the last 2.1. two decades, information on the molecular characteristics of desaturases has amassed rapidly; however, information on the Female of O. scapulalis were collected at Matsudo, Japan, other enzymes involved in pheromone biosynthesis is quite and individually housed in a plastic cup to allow oviposition. The limited. offspring were reared on an artificial diet (SilkmateÔ 2S, Nosan The molecular characterization of FAR was first achieved by Corp., Yokohama, Japan) as previously described (Takanashi et al., Moto et al. (2003), who succeeded in identifying the pheromone- 2005). Pupae were sexed, and female pupae were maintained at gland-specific FAR (pgFAR) in Bombyx mori (Matsumoto et al., 23.0 2.0 C under a 16-h light: 8-h dark photoperiod. Emerged 2007). Although a report on the molecular characterization of female moths were collected daily during mid scotophase. Females 2–4 days old were used for experiments. In terms of pheromone production type, females of this natural population are a mixture of * Corresponding author. Tel.: þ81 3 5841 1851; fax: þ81 3 5841 5061. E-type, Z-type and their hybrids, the hybrids being the most E-mail address: [email protected] (Y. Ishikawa). abundant (Takanashi et al., 2005). E-type, Z-type and hybrids

0965-1748/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.10.008 B. Antony et al. / Insect Biochemistry and Molecular Biology 39 (2009) 90–95 91 produce a 99:1, 3:97, and 64:36 mixture of E11-14:OAc and Table 1 Z11-14:OAc, respectively (Huang et al., 2002). Primers used for degenerate PCR and RACE experiments of FAR-XIII. Name Sequence (50–30) þ 2.2. Poly(A) RNA isolation and cDNA synthesis Degenerate primers Os_F1_deg ACNGGHTTYMTDGGVAA Poly(A)þ RNA (mRNA) was purified from the pheromone gland Os_F2_deg YAYRTDTCBACWGCHTA of females using a FastTrack MAG mRNA isolation kit (Invitrogen, Os_R2_deg RTADGCWGTVGAHAYRT FAR_Gen_R GMTTTKGTGTANGYRTAYGTRTTHGG Carlsbad, CA) as follows. The terminal abdominal segments (TAS) of FAR_LAT_F ACNGGNGSNACNGGNTT female moths were extruded by applying gentle pressure to the FAR_LAT_Rev_TCN RTANGCNGTNGANACRT abdomen, and cut with micro-scissors. The TAS (n ¼ 60) were FAR_LAT_Rev_AGY RTANGCNGTRCTNACRT washed in insect ringer 3 times and then cut at the level of the RACE Primers lateral apophysis on one side to open the TAS. Fat bodies and other FARXIII30F1 AGCAGACAGAAGACCTGACCATTGAAGC inner tissues were removed from the TAS as thoroughly as possible FARXIII30F2 CAGAAGACCTGACCATTGAAGCGA FARXIII50R1 CCTCGCTTCAATGGTCAGGTCTTC with a fine needle. Then the 8th–9th intersegmental membranes, 0 FARXIII5 R2 GCTTCAATGGTCAGGTCTTCTGTCTGCT which include pheromone-producing cells, were incubated at 37 C FARXIII_30W_I GTCTCTAGCCGATACAGTAATG for 30 min in 250 ml of insect ringer with 1% trypsin and 2.5 mlof FARXIII_30W_II GGCAACAAAGGAGTCAAGG RNase inhibitor (40 U/ml, Takara Bio Inc., Ohtsu, Japan) to separate FARXIII_30W_III CGATGTCACTGAAATTGAGTGG pheromone-producing cells from the cuticle. After cooling to room FARXIIIFL_F1 CAGTCTAGAAAAATGTCAGC FARXIIIFL_R1b GCTCAAAAACTACTGGACCG temperature, it was centrifuged at 3000 g for 5 min and then washed 2 times in the above solution without trypsin. After centrifugation, 100 ml of lysis buffer L4 (FastTrack MAG mRNA the WizardÒ SV Gel and PCR Clean-up system (Promega, Madison, isolation kit, Invitrogen) was added and digested tissues were WI), and ligated into a pGEMÒ-T easy vector (Promega), and passed through a 23-gauge needle, and thereafter mRNA was iso- transformed into Escherichia coli competent cells (JM109, Takara lated by following the manufacturer’s protocol, with the addition of Bio). Recombinant clones were amplified with M13 universal DNase (Qiagen, Valencia, CA) treatment. primers, and sequenced in both directions using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) on an 2.3. cDNA normalization ABI PRISM 310 Genetic Analyzer (Applied Biosystems). In parallel to the effort to obtain FAR fragments by degenerate The first-strand cDNA was prepared from mRNA extracted from PCR, a cDNA library was constructed and screened randomly. The the TAS of female O. scapulalis using a SMARTÔ PCR cDNA library normalized cDNA was directly ligated into a pGEMÒ-T easy vector construction kit (Clontech, Mountain View, CA) following the following the manufacturer’s protocol, and transformed into E. coli manufacturer’s instructions except that the CDS-3M adaptor cells. Recombinant clones were subjected to PCR using the provided in the Trimmer Direct kit (Evrogen, Moscow, Russia) was universal primers. The products of 1–2 kb were purified using the used instead of the CDSIII 30 PCR primer. The cDNA was precipitated WizardÒ SV Gel and PCR Clean-up system, and sequenced. with ethanol and dissolved in RNase-free water to a final concen- tration of 100 ng/ml. The amplification of the first-strand cDNA and 2.5. Sequence data analysis duplex-specific nuclease treatment were carried out following the manufacturer’s protocol. PCR was performed using an Advantage 2 Partial gene sequences obtained were joined using BioEdit PCR kit with a 50 PCR primer (Clontech) under the following (BioEdit, North Carolina State University, USA), and aligned using conditions: 18 cycles of 95 C for 7 s, 66 C for 30 s, and 72 C for the Clustal W program with the sequence of B. mori pgFAR (Moto 6 min. The first and second amplifications of the normalized cDNA et al., 2003), and sequences of the , mouse and jojoba FARs. were performed with the Trimmer direct PCR primers M1 and M2 Genealogical analysis of FAR genes was carried out using neighbor- under the above conditions for 18 and 12 thermal cycles, respec- joining method with TREECON for windows version 1.3b (Univer- tively. After Proteinase K treatment, SfiI digestion, and purification sity of Konstanz, Germany) with jojoba FAR (JJFAR) (Metz et al., with a chroma spin column (Clontech), the normalized cDNA was 2000) as an outgroup. ethanol precipitated and dissolved in PCR water (Advantage 2 PCR kit, Clontech) to a concentration of 100 ng/ml. The normalized cDNA 2.6. Tissue specificity studies by RT-PCR was checked for the presence of the D11-desaturase gene by PCR using desL1/desR1 primers (Fukuzawa et al., 2006). Total RNA was isolated from the whole head, thorax, and abdomen (with and without TAS) of 3-day-old O. scapulalis female 2.4. Degenerate PCR amplification and cloning moths (n ¼ 10), and whole head, thorax, and abdomen of male moths (n ¼ 10) using an RNeasy mini kit (Qiagen) with DNase Degenerate primers for the amplification of FAR genes were treatment. The first-strand cDNA was synthesized from 1 mg of total designed (Table 1) based on the published amino acid sequences of RNA using an RNA PCR kit (AMV) with an oligo-dT adaptor primer FAR proteins of B. mori (GenBank accession No. AB104896), (Takara Bio). Touch down PCR was performed using primer sets (AY600449 and BC022267), mice (BC007178 and BC055759) and designed for the specific amplification of each FAR-like sequence the jojoba plant (Simmondsia chinensis; AF149917). Touch down (Table 2). The conditions were 94 C for 1 min, 10 cycles of 94 C for PCR amplifications were performed using different combinations of 30 s, 60 C for 1 min (lowered by 1 C/cycle), and 72 C for 1 min forward and reverse primers (Table 1), under the following condi- followed by 20 cycles of 94 C for 30 s, 50 C for 1 min and 72 C for tions: 94 C for 1 min, 12 cycles of 94 C for 30 s, 52 C for 1 min 1 min, and a final extension at 72 C for 7 min. The PCR products (lowered by 1 C/cycle), and 72 C for 1 min followed by 30 cycles of were separated by 2% agarose gel electrophoresis. 94 C for 30 s, 40 C for 1 min and 72 C for 1 min, and a final extension at 72 C for 10 min. In addition, low annealing temper- 2.7. Rapid amplification of cDNA ends (50 and 30 RACE) ature PCR (55 cycles of 94 C for 30 s, 35 C for 1 min and 72 C for 1 min) was carried out using the last three degenerate primer To obtain a full-length cDNA, 30 and 50 RACE were performed combinations (Table 1). The band of expected size was purified with using the GeneRacerÔ kit (Invitrogen) according to the Download English Version: https://daneshyari.com/en/article/1982697

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