Insect Science (2012) 19, 55–63, DOI 10.1111/j.1744-7917.2011.01427.x ORIGINAL ARTICLE Genome-wide screening and transcriptional profile analysis of desaturase genes in the European corn borer moth Bingye Xue1,†, Alejandro P. Rooney2 and Wendell L. Roelofs1 1Department of Entomology, NYSAES-Cornell University, Geneva, NY, 2Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL, USA Abstract Acyl-coenzyme A (Acyl-CoA) desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on the basis of biochemical functionality and phylogenetic affinity. In this study both copy numbers and transcriptional levels of desaturase genes in the European corn borer (ECB), Ostrinia nubilalis, were investigated. The results from genome-wide screening of ECB bacterial artificial chromosome (BAC) library indicated there are many copies of some desaturase genes in the genome. An open reading frame (ORF) has been isolated for the novel desaturase gene ECB ezi-11β from ECB gland complementary DNA and its functionality has been analyzed by two yeast expression systems. No functional activities have been detected for it. The expression levels of the four desaturase genes both in the pheromone gland and fat body of ECB and Asian corn borer (ACB), O. furnacalis, were determined by real-time polymerase chain reaction. In the ECB gland, 11 is the most abundant, although the amount of 14 is also considerable. In the ACB gland, 14 is the most abundant and is 100 times more abundant than all the other three combined. The results from the analysis of evolution of desaturase gene transcription in the ECB, ACB and other moths indicate that the pattern of 11 gene transcription is significantly different from the transcriptional patterns of other desaturase genes and this difference is tied to the underlying nucleotide composition bias of the genome. Key words bacterial artificial chromosome (BAC) library, desaturase, European corn borer, G+C, sex pheromone Introduction fatty acids that generate a blend of pheromones unique to each species. Desaturase genes are encoded by a large Acyl-coenzyme A (Acyl-CoA) desaturases play a key role multigene family. A considerable amount of research has in the biosynthesis of female moth sex pheromones. They been conducted on the structure, function and evolution are responsible for the production of distinct, desaturated of moth sex pheromone desaturase genes (e.g., Knipple et al., 1998, 2002; Liu et al., 1999, 2002a,b, 2004; Rosen- field et al., 2001; Hao et al., 2002; Roelofs et al., 2002; Correspondence: Wendell L. Roelofs, Department of Ento- Xue et al., 2007; Lassance & Lofstedt,¨ 2009), and they mology, New York State Agricultural Experiment Station, Cor- have been divided into five subgroups on the basis of bio- nell University, 630 West North Street, Geneva, New York, NY chemical functionality and phylogenetic affinity (Roelofs 14456, USA. Tel: +1 315 789 1426; fax: +1 315 787 2326; et al., 2002; Roelofs & Rooney, 2003; Liu et al., 2004; email: [email protected] Xue et al., 2007; Rooney, 2009). †Current address: Department of Plant Pathology, North Car- Previously, we characterized a novel class of desaturase- olina State University, Raleigh, NC 27607, USA retroposon fusion genes from the European corn borer C 2011 The Authors 55 Journal compilation C Institute of Zoology, Chinese Academy of Sciences 56 B. Xue et al. (ECB), Ostrinia nubilalis, and the Asian corn borer cDNA cloning and functional assay for ECB ezi-11β (ACB), Ostrinia furnacalis (Xue et al., 2007). The novel and ACB ezi-11β desaturase genes indicate that the evolution of moth sex pheromone desaturases is much more complex than pre- Complementary DNAs (cDNAs) were made from viously recognized due to the gene duplication and retro- pheromone gland and fat body of both ECB and ACB poson invasion to the genome. Thus, questions arise on (Roelofs et al., 2002). Nested PCRs were used for whether these novel desaturase genes are transcribed and cDNA cloning of ECB ezi-11β and ACB ezi-11β functional, and how these genes are distributed in the ECB by two sets of primers designed from the genomic genome. DNA sequences (GenBank accession nos. EF113395, Another more interesting scenario in the study of the EF113393). The primers are ECB-E/Z11–2-F1 (5- structure, function and evolution of moth sex pheromone CTAATATAAACATGGCCGAAAT-3) and ECB-E/Z11– desaturases is the pattern of reciprocal gene activation– 2-R1 (5-CTTGTTCAATAAATTATAAT-3) and ECB- deactivation observed in ECB and ACB. The female E/Z11–2-F2 (5-ACATGGCCGAAATACCAAC-3) and ECB pheromone gland contains four different desaturase ECB-E/Z11–2-R2 (5-TAATTTAGACTTCATTCTAC- gene messenger RNA (mRNA) transcripts (11, 14, 3). One fragment (GenBank accession no. FJ906800) 9 [16>18] and 9 [18>16]), but only the 11 gene was isolated from the ECB gland cDNA and the open is expressed to produce an enzyme that functions in fe- reading frame (ORF) of 966 bp for the ECB ezi-11β male sex pheromone biosynthesis (Roelofs et al., 2002). gene was deduced from this fragment and ligated into Likewise, the 11, 14, 9 (16>18) and 9 (18>16) YEpOLEX (Knipple et al., 1998; Liu et al., 1999) and genes are also transcribed in the female ACB pheromone pYES2 (Toke & Martin, 1996; Liu et al., 2002a,b) gland, but in this case it is the 14 gene that is ex- expression vectors and transformed to yeast ole1 and pressed to produce an enzyme that functions in female Elo1 cells. sex pheromone biosynthesis. This odd pattern was diffi- Functionality was subsequently analyzed by mass spec- cult to explain until Lassance & Lofstedt¨ (2009) showed tral analysis of DMDS (Dimethyl disulfide) adducts. that the ECB 14 gene is expressed in the male hair- pencil tissue where it functions in the male sex pheromone biosynthetic pathway. Thus, one might speculate that the Quantitative real-time PCR 14 gene product would be found in the ACB male hair pencil tissue as well, but it is not (Lassance & Lofstedt,¨ To determine the expression level of desaturase genes, 2009). These paradoxical observations lead one to wonder mRNAs were isolated from 2–3-day-old ECB and ACB how this complex system of desaturase gene expression female glands and fat-body and reverse-transcribed into evolved. cDNA. A real-time PCR system, Cepheid’s Smart Cycler In the present paper, we report the results of ECB System (Cepheid, Inc., Sunnyvale, CA, US) was used for Bacterial Artificial Chromosome (BAC) library screen- the amplification of target cDNAs. Reactions were car- ing, novel gene functional analysis, and the analy- ried out in a total volume of 25 μL/tube. Each reaction sis of desaturase gene transcriptional patterns in the master mixture contained 3 mmol/L MgCl2,0.1μmol/L fat body and pheromone gland of the ECB and of each primer, 0.2 mmol/L deoxynucleotide triphos- ACB. phate (dNTP), 0.5 μL10× SYBR Green I, 1.25 U Hot Start Ex Taq (TaKaRa Bio USA, Madison, WI, USA), 2.5 μL10× buffer, 5 μL template DNA and water. In Materials and methods every run, one negative control (water), seven standard cDNA, and eight tissue samples including two replica- ECB BAC library screening tions of ECB gland and fat body cDNA, ACB gland and fat body cDNA, were included. The following protocol European corn borer BAC library was constructed was used: initial denaturation for 2 min at 95◦C followed by Clemson University Genomics Institute (CUGI). by 45 cycles of: (i) 15 s at 95◦C; (ii) 20 s at 60◦C; and (iii) Primers were designed based on the sequences (Table 1) 20 s at 72◦C. The reaction was heated above the melting deposited in the GenBank and polymerase chain reaction temperature of the dimers for each gene for an additional (PCR) products labeled with 32P were used as probes. 10s and fluorescence measurements were made at the end Hybridization followed the protocol provide by CUGI of the additional extension phase. The threshold line was (http://www.genome.clemson.edu/resources/protocols). set at 30 fluorescent units on the Y-axis and the primary The screening procedure is summarized in Fig. 1. curve was selected in the curve analysis. C 2011 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 19, 55–63 European corn borer pheromone desaturase evolution 57 Table 1 Polymerase chain reaction (PCR) primers for making probes to screen European corn borer bacterial artificial chromosome (ECB BAC) library. Name of genes PCR product size (bp) PCR primer sequence Accession no. OnuZ/E11 309 bp 5-AATTATATTTTATTTTTTTGCTGAG-3 AF441221 5-CTATATAAATCAGCCACATCACAC-3 OnuZ/E14 211 bp 5-CAGACATAGACGCCACCAACCCT-3 AF441220 5-GACGCGATTTTTACATTTCCTCC-3 OnuZ9(16>18) (Z9–1) 208 bp 5-CTCCTAATATTAAGGACGGAGCT -3 AF243047 5-TGCCATTTCGCTGAAAACAGGAAT-3 OnuZ9(18>16) (Z9–2) 194 bp 5-TGCCGCCACAAGGTGCAGAGAG-3 AF430246 5-CATCACAGTTGTGAGCATCAGG-3 ECB-ezi-11α 266 bp 5-TCTATACTCGGAATTACTGCAGCTGCT-3 EF113398 5-GATTTGCCTTGTCGTTTCACCTCTTCG-3 ECB-ezi-11β 303 bp 5-TATGTATCACATAACTATAATAGGC-3 EF113395 5-TTGTATAAGTCGGACATGTCCATAGAC-3 ECB-ezi-11ψ 335 bp 5-AGTGCTGATGCATATATTGGGTACC-3 EF113404 5-CATTCTGGAGGCGTATGACAGGA-3 A specific primer pair (Table 2) was used to determine ECB-ezi-11β, ECB-11ψ (Fig. 1, Table S1). Further the transcription level for each of the four desaturase analysis by colony PCR and DNA sequencing confirmed genes in ECB and ACB. Melting curve analyses were that there were two distinct clones of ECB-11 (75M1, performed immediately following PCR to identify spe- 2I23), 8 clones of ECB-ezi-11α and ECB-ezi-11β cific PCR products and primer dimers as the temperature (32A7, 32H12, 51K12, 76E13, 76N10, 79K15, 32G3, slowly increased from 60 to 95◦C.