Two conserved Z9-octadecanoic acid desaturases in the red flour beetle, Tribolium castaneum

Gene 468 (2010) 41–47

Contents lists available at ScienceDirect

Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e

Methods paper

Two conserved Z9-octadecanoic acid desaturases in the red flour beetle, Tribolium castaneum Irene Horne, Nerida Gibb, Katherine Damcevski, Karen Glover, Victoria S. Haritos ⁎ CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia

a r t i c l e

i n f o

Article history: Accepted 4 August 2010 Available online 13 August 2010 Received by J.G. Zhang Keywords: Acyl-coenzyme A desaturase Coleoptera Z9-octadecanoic acid Gene duplication ole1 complementation Developmental stage

a b s t r a c t Z9 Desaturases catalyse the formation of a cis-unsaturated bond in the Δ9 position of the saturated fatty acids stearate and palmitate. They are considered essential enzymes in eukaryotic organisms as their Z9 unsaturated fatty acid products are required for homeostatic roles such as maintenance of membrane fluidity. Two putative Z9 acyl Coenzyme-A desaturase genes were identified in the red flour beetle, Tribolium castaneum, genome (TcasZ9desA and B) based on their similarity to acyl CoA-desaturases of other insects. TcasZ9desA and B share 75% nucleic acid sequence identity and appear to be functionally conserved; the genes were cloned and expressed in the yeast strain Saccharomyces cerevisiae (ole1); both genes complemented the yeast requirement for Z9 fatty acids and produced substantial quantities of Z9 desaturated products with a stearate N palmitate chain length preference. Quantitative PCR analysis of transcripts in RNA obtained from adult, larval and pupal stages of the beetles show TcasZ9desA and B are expressed at similar levels in all stages, with the pupal stage having the lowest expression. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction The endogenous production of Z9 fatty acids is vital in eukaryotes for maintaining correct cell membrane fluidity (Tiku et al., 1996) and for other homeostatic roles including as a precursor to polyunsaturated fatty acid and pheromone syntheses. Eukaryotes possess at least one essential and conserved fatty acid desaturase which introduces a double bond in the Z (cis) orientation at the Δ9 position of the 18carbon chain length saturated fatty acid, stearate, or the 16-carbon homologue palmitate. In insects, Z9 acyl CoA desaturase gene expression has been shown to be responsive to temperature and feeding following starvation. In the onion maggot, Delia antiqua, Z9 desaturase expression was upregulated in pupae in response to cold (Kayukawa et al., 2007) resulting in increased cold tolerance and content of unsaturated fatty acid in brain phospholipids. Further, Riddervold et al. (2002) showed significantly increased Z9 desaturase expression in response to feeding in newly eclosed adult crickets following a period of starvation. Over recent years there has been intensive investigation of acylCoA desaturases from insects among moths, flies and crickets

that perform homeostatic and specialist roles such as pheromone synthesis (Dallerac et al., 2000; Eigenheer et al., 2002; Labeur et al., 2002; Liu et al., 2004; Park et al., 2008; Riddervold et al., 2002; Rosenfield et al., 2001; Xue et al., 2007; Zhou et al., 2008). However, the conserved Z9 desaturases from Coleoptera (beetles), the largest order of eukaryotic organisms, have not been isolated and functionally characterised. Tribolium castaneum, the red flour beetle, is a welldeveloped model in developmental genetics and an economically important stored grain pest. The genome of T. castaneum has been sequenced and will play an important role in understanding the molecular physiology of this beetle. Here, we describe the cloning and functional characterisations of two conserved Z9 acylCoA desaturases from T. castaneum and examine their gene expression across developmental stages. Identification of two apparently homeostatic Z9 desaturases with the same activity was unexpected especially with the finding that they are expressed at similar levels in each developmental stage. 2. Materials and methods 2.1. Gene prediction

Abbreviations: BLOSUM, Blocks of amino acid substitution matrix; CoA, Coenzyme A; Cq, quantification cycle; CSIRO, Commonwealth Scientific and Industrial Research Organisation; DMOX, dimethyloxazoline; dN/dS, nonsynonymous to synonymous ratio; FAME, fatty acid methyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEGA, Molecular Evolutionary Genetics Analysis; qPCR, quantitative PCR. ⁎ Corresponding author. Current address: CSIRO Entomology, 343 Royal Pde, Parkville, 3052, Australia. Tel.: + 61 3 9662 7235. E-mail address: [email protected] (V.S. Haritos).

All full-length insect desaturase protein sequences (obtained from the Pfam database—http://www.sanger.ac.uk/Software/Pfam/ index.shtml) were aligned using the Clustal X program (Thompson et al., 1997). From this sequence alignment, a highly conserved sequence after the third histidine box was observed (HNYHHAYPWDYKAAEIGMPLNSTASLIRLCASLGWAYDLKSV) and used in a tBLASTn

0378-1119/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.08.003

42

I. Horne et al. / Gene 468 (2010) 41–47

analysis (Altschul et al., 1997) against the T. castaneum genome (http://www.bioinformatics.ksu.edu/BeetleBase/; Tribolium genome sequencing consortium, 2008). From this analysis, 15 putative desaturase sequences were obtained. Each of the contigs was then subjected to a gene prediction program in Softberry (http://www. softberry.com/cgi-bin/programs/gfin/fgenesh) using parameters for Brugia malaya and Caenorhabditis elegans.

2.2. Amplification of desaturases Adult T. castaneum TC4 strain were obtained from long term cultures at CSIRO Entomology reared on wholemeal flour with brewers yeast, at 25 °C and 55% relative humidity, in the dark. Total RNA was extracted from adult beetles using the Trizol® reagent according to the manufacturer's instructions (Invitrogen Corporation, Carlsbad, CA, USA) and further purified using the RNeasy mini protocol for RNA cleanup (QIAgen, Valencia, CA, USA). Two desaturase genes with high homology to other insect Z9 desaturases, TcasZ9desA and B, were amplified from 37 ng of T. castaneum total RNA using the SuperscriptIII RT/Platinum Taq DNA polymerase kit (Invitrogen) according to the manufacturer's instructions. TcasZ9desA was amplified using the primers Z9desAfor (5′ GGATCCATGCCACCCTATGTGTCC) and Z9desArev (5′GCATGCTTAATTAAAATCGTCAGA) containing a BamHI site and an SphI site, respectively (underlined). TcasZ9desB was amplified using the Z9desBfor (5′ GGATCCATGACACCAAATGCTTCA) and Z9desBrev (5′GAATTCCTACGCACTCTTCCTATG) containing a BamHI site and an EcoRI site, respectively (underlined). Approximately 1 kb amplicons were obtained from each RT-PCR and purified using the QIAquick PCR purification kit (QIAgen). The TcasZ9desA and B-containing fragments were cloned into pGEM T Easy (Promega), to generate pGEM-TcasZ9desA and pGEM-TcasZ9desB, respectively, and the sequences confirmed. BLASTn analysis was conducted with TcasZ9desA and B against the nucleotide collection in Genbank.

2.3. Sequence and phylogenetic analysis Deduced amino acid (aa) sequences for TcasZ9desA and B and characterised Z9 desaturases from insects were aligned using ClustalW (Thompson et al., 1994) and their sequence identities/ similarities were calculated using the BLOSUM62 similarity matrix. BLASTp analyses (Altschul et al., 1997) of TcasZ9desA and TcasZ9desB proteins were conducted against the non-redundant protein database of NCBI. Predictions for transmembrane regions in the T. castanuem sequences were made using TMpred (Hofmann and Stoffel, 1993). The non-synonymous to synonymous changes and transition/transversion ratio were determined using DIVERGE (Accelrys-GCG). Phylogenetic analyses were conducted on a wide range of insect desaturases with representatives from five insect orders and activities that included Δ9, Δ10–13, Δ12 and Δ14 desaturation. Protein sequences were aligned using ClustalW (Thompson et al., 1994) and then a phylogenetic tree was constructed using the neighbour-joining method of Saitou and Nei (1987) using the package MEGA version 4 (Tamura et al., 2007). Bootstrap analysis with 1500 replicates was carried out to determine statistical support for tree branches. The insect orders and full species name of organisms whose protein sequences were used in the analysis are as follows: Orthoptera: Acheta domesticus Diptera: Delia antiqua, Drosophila melanogaster, Musca domestica, Anopheles gambiae Lepidoptera: Bombyx mori, Choristoneura parallela, Epiphyas postvittana, Helicoverpa zea, Mamestra brassicae, Manduca sexta, Ostrinia nubilalis, Planototrix octo, Spodoptera littoralis, Thaumetopoea pityocampa, Trichoplusia ni Hymenoptera: Apis mellifera Coleoptera: Diaprepes abbreviatus, Tribolium castaneum (TcasZ9desA HM234671 and TcasZ9desB HM234672).

2.4. Yeast expression of TcasZ9desA and TcasZ9desB The pGEM T Easy clones, pGEM-TcasZ9desA and pGEM-TcasZ9desB were digested with BamHI–SphI and BamHI–EcoRI, respectively. The desaturase-encoding fragments were ligated with similarlydigested pYES2 (Invitrogen Corporation, yeast expression plasmid) to create pYES-TcasZ9desA and pYES-TcasZ9desB, respectively. Positive clones were determined by restriction analysis and retained for yeast transformation. The pYES-TcasZ9desA and pYES-TcasZ9desB were transformed into Saccharomyces cerevisiae ole1 strain (MATa, ole1Δ:LEU2, leu2-3, leu2-112, trp1-1, ura3-52, his4) using the Sigma Yeast Transformation kit. Transformants were selected on minimal media lacking uracil (Kaiser et al., 1994) and containing 1 mM cis-10heptadecenoic acid and 1% tergitol (NP-40). To test for complementation of the OLE1 phenotype, transformants were patched onto YP medium (20 g l−1 peptone, 10 g l−1 yeast extract) containing 2% galactose. A control transformant, containing pYES2, was also prepared and tested. To examine the production of desaturase products, S. cerevisiae OLE1/pYES-TcasZ9desA and OLE1/pYES-TcasZ9desB and control OLE1/pYES were cultured in 10 ml minimal medium without uracil, containing 2% galactose to induce gene transcription, 1% raffinose, and 0.5 mM Z-11-heptadecenoic acid added as a 1% tergitol solution (for OLE1/pYES control). The induced culture was grown for 72 h at 20 °C with shaking. Cells were collected by centrifugation (2000 g) and stored at −20 °C. 2.5. Lipid analysis of recombinant yeast and whole adult insects Yeast cells were washed sequentially with 1% tergitol and MilliQ water with centrifugation at 1500g for 5 min at + 4 °C and dried in a Savant SpeedVac Plus SC110A concentrator/dryer. Cells were directly treated with methanol/hydrochloric acid/chloroform (10:1:1) in a sealed test tube with heating at 90 °C for 60 min to convert lipids to fatty acid methyl esters (FAME). When cool, saline and hexane were added with shaking, and the hexane layer containing FAME was transferred to a vial for analysis by Varian 3800 gas chromatograph fitted with a BPX70 capillary column (Phenomenex 30 m × 0.32 mm × 0.25 μm). Injections were made in the split mode using helium as the carrier gas and an initial column temperature of 60 °C, raised at 20 °C/ min until 170 °C, held for 5 min, then raised at 50 °C/min until 255 °C. Confirmation of FAME and dimethyloxazoline (DMOX) derivatives was obtained using a Varian 3800 gas chromatograph/1200 single quadrupole mass spectrometer. Mass spectra were acquired under positive electron impact in full scan mode between 50 and 400 amu at the rate of 2 scans per sec. Confirmation of the identity of a fatty acid was achieved by the conversion of the fatty acid to its DMOX derivative using the method of Yu et al. (1988) and comparison with DMOX mass spectra described in Dobson and Christie (2002) and references within. 2.6. Quantitative PCR analysis of TcasZ9desA and TcasZ9desB in various life stages Total RNA was isolated from separately pooled larvae, pupae and adult T. castaneum lifestages using a RiboPure Kit (Ambion, Foster City, CA, USA) and quantified using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA was DNase treated using SV Total RNA Isolation System (Promega, Madison, WI, USA). cDNA was then generated from 1 μg of total RNA using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). A no-reverse transcription control was included to test for DNA contamination. Purified RNA was visualized using electrophoresis and quantified. The ratio at 260/280 nm for the RNA samples was 2.0 and the ratio at 230/ 260 nm was 2.3.

I. Horne et al. / Gene 468 (2010) 41–47 Table 1 Summary of forward and reverse primer sequences used in the quantitative PCR amplifications for TcasZ9desA and B. Gene (accession no.)

Forward primer 5′–3′

Reverse primer 5′–3′

GAPDH XM_969088.1 Tubulin XM_961399.1 TcasZ9desA HM234671 TcasZ9desB HM234672

GGATACGCACTCGTCGATTT

TCAACAACTCGGCTCGAATACC

GAAGCTCGTGAAGATTTGGC

ACCTTCGCCTTCTCCTTCTC

GAACTGGTGATGGGTCGC

AATCGCCCCTTCATAATCCT

TCAGTCTCAGGAAGACTACCAAG

CATAAGTTACCTAACAATCAGTC

obtained. The inbuilt software in CFX96 Real Time System (Bio-Rad,) was utilized to calculate gene expression data and gene stability values for the reference genes tubulin and GAPDH. A gene stability value (M) of 0.3116 was obtained within the acceptable range for stability for heterogeneous tissues (Hellemans et al., 2007). 3. Results and discussion 3.1. Identification of Z9 desaturases in the T. castaneum genome and sequence analysis

Table 2 Amino acid sequence identities (%) between Z9 and Z12 desaturase sequences from T. castaneum (TcasZ9desA and B, TcasZ12des) and characterised desaturase representatives from other insects. Sequence (accession)

TcasZ9desA

TcasZ9desB

TcasZ9desA (HM234671) TcasZ9desB (HM234672) AdomZ9des (AF338465) HzeaZ9des (AF272343) DmelZ9des (AJ245747) MdomZ9des (AF417841) TcasZ12desa (EU159449)

– 80.1 66.0 65.4 62.4 60.7 47.9

80.1 – 63.8 64.3 60.6 60.3 46.3

a

43

EU159449 Zhou et al. (2008).

Primers for the quantification of TcasZ9desA and B and housekeeping genes GAPDH and tubulin were designed to amplify from the 3′end of the ORF into the 3′UTR using the program Primer-BLAST (http://www.ncbi.nlm.nih.gov) and are given in Table 1. Secondary structure predictions for the amplicons were assessed using the mfold program (http://www.bioinfo.rpi.edu/, Zuker, 2003). Primers were tested against T. castaneum cDNA using Taq polymerase (Invitrogen Corporation). Amplification products were run on a 1.3% agarose gel in TAE to check primer size and dimer formation, and ligated into pCR2.1-TOPO (Invitrogen Corporation) for subsequent sequence verification. All genes tested amplified single products and the derived sequences were identical to the original sequences. Quantitative PCR (qPCR) reactions for each gene were optimized by performing gradient PCR with primer dilutions using IQ SYBR Green Supermix ) and CFX96 Real Time System both supplied by BioRad,). The combination of annealing temperature and primer concentration giving the lowest Cq was then used for subsequent qPCR experiments. Five-fold serial dilutions of the cDNA were analysed in triplicate to create reaction efficiency curves; correlation coefficients achieved ranged between 0.98 and 1.0. Controls without added template were also included. Reaction efficiencies of 83% (GAPDH and TcasZ9desB), 85% (TcasZ9desB) and 89% (tubulin) were

57 bp intron

5’ UTR 213 bp

-65 TATATAAA

Fifteen putative desaturases were identified from the T. castaneum genome using a homology search with the conserved domain to identify desaturase-containing contigs. It was assumed that the T. castaneum desaturases with highest sequence conservation to Z9 desaturases of other insects would be the most likely candidate(s). Two T. castaneum genes had N60% sequence identity to other homeostatic insect Z9 desaturases based on a comparison of their encoded protein sequences and were designated TcasZ9desA and B (Table 2). The next closest translated T. castaneum putative desaturase gene had substantially lower aa sequence identity (b55%) to homeostatic Z9 desaturases. Although it is likely that TcasZ9desA and B resulted from gene duplication, it is not clear at this stage whether they are co-located in the genome. TcasZ9desA (LOC658103) is found on chromosome LG10 but the location of TcasZ9desB (LOC657393) is currently unknown. BLASTn analysis of TcasZ9desA gave 100% match to the nucleotides 48– 1109 inclusive of XM_964514.2 and TcasZ9desB returned hits to two variants of the same gene: variant 1 (XM_961869; match between 91 and 1143) and variant 2 (XM_961869; match between 111 and 1163) which differ in 5′UTR sequence. The sequence upstream of the predicted ATG start sites for TcasZ9desA and B were examined for promoter and coding regions. Intron and exon sites were obtained from FGENESH analysis of the relevant genomic sequences and these features are summarised in Fig. 1. The location of the single intron in the coding regions of TcasZ9desA and B is in a conserved position found in all known intron-containing insect desaturases except the cricket Z9 desaturase (Riddervold et al., 2002; Rosenfield et al., 2001) but it lacks introns located at the sites encoded by QKK and PYDK (relative position indicated in Fig. 1) observed in other desaturase genes. The encoded protein sequences for TcasZ9desA and B contained three histidine boxes (dashed lines, Fig. 2) which are involved in ironbinding, including eight histidines shown to be catalytically essential in other desaturases (Shanklin et al., 1994) and three other aa motifs that are common to insect Z9 desaturases: FFSHI/VGW, QKKYY and PWDY (open boxes, Fig. 2). Excluding the N-terminal region, the conservation of aa sequence among the desaturases shown in Fig. 2 is almost complete albeit the last common ancestor of these species was ~400 Myr ago (Grimaldi and Engel, 2005) confirming their likely conserved function. The N-terminus of Z9 desaturases can be highly

ATG

846 bp

Exon 1

E Exon 2

Q QKK

213 bp ATG

1570 bp intron

TcZ9desA

837 bp

-1712 TATATAAA

PWDY

Exon 1

Exon 2

QKK

PWDY

TcZ9desB

324 bp intron

Fig. 1. Genomic arrangement of TcasZ9desA and B and upstream regions showing the location of promoters, and intron and exon size and number for the two genes.

44

I. Horne et al. / Gene 468 (2010) 41–47

10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|

TcasZ9desA TcasZ9desB DmelZ9des MdomZ9des AdomZ9des HzeaZ9des

MPPY-----------------------------VSHVTGVLDEND---EEVST---KNILPEVTKP-ENR MTPN-----------------------------ASIPTGVLHEND---EEVSN---ATLPPEVNKP-DDR MPPNAQAGAQSISDSLIAAASAAADAGQSPTKLQEDSTGVLFECD---VETTDGGLVKDITVMKKA-EKR MPPNAAPPTPALTESLLASANID---GANPKVLHEATTGVLFEQD---AETIDGGLVKDIERLKKA-EKR MAPN----------------------------ITSAPTGVLFEGDTIGPAAKDQQAEVNAPEAKKPREPY MAPN----------------------------ISEDVNGVLFESD---AATPD--LALSTPPVQKA-DNR 80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|

TcasZ9desA TcasZ9desB DmelZ9des MdomZ9des AdomZ9des HzeaZ9des

KLQLVWRNIILFAYLYLASFYGLYLMFTSAKLATSIFAYFLYQLGGFGITAGAHRLWAHRSFKAKWPLRL KLQLVWRNIILFAYLHLAALYGIWIMFTSAKVATSLFGILLYQLGGFGITAGAHRLWAHRSYKAKWPLRL RLKLVWRNIIAFGYLHLAALYGAYLMVTSAKWQTCILAYFLYVISGLGITAGAHRLWAHRSYKAKWPLRV KLKLVWRNIIAFGYLHLAALYGAYLMFTSAKWQTIVFAFALYVVSGLGITAGAHRLWAHRSYKAKWPLRL RRQIVWRNVILFIYLHLAALYGAYLAFTSAKIATTIFAIILYQVSGVGITGGAHRLWAHRSYKAKWPLRV PKQLVWRNILLFAYLHLAALYGGYLFLFSAKWQTDIFAYILYVISGLGITAGAHRLWAHKSYKAKWPLRV 150 160 170 180 190 200 210 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|

TcasZ9desA TcasZ9desB DmelZ9des MdomZ9des AdomZ9des HzeaZ9des

LLAFCNTLAFEDSVIDWSRDHRVHHKFSETDADPYNAKRGFFFSHIGWLLCRKHPQVKEKGKQIDLSDLY ILTFCNTLAFEDSVIDWSRDHRVHHKFSETDADPHNAKRGFFFSHVGWLLCRKHPQVKEKGKQIDLSDLY ILVIFNTIAFQDAAYHWARDHRVHHKYSETDADPHNATRGFFFSHVGWLLCKKHPEVKAKGKGVDLSDLR ILMIFNTIAFQDAAYHWARDHRVHHKYSETDADPHNATRGFFFSHIGWLLCKKHPEVKAKGKGVDLSDLK ILMLCNTLAFQNHIYEWARDHRVHHKFSETDADPHNATRGFFFSHVGWLLVRKHPDVKEKGKGIDMHDLE ILVIFNTVAFQDAAMDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPDLKEKGKGLDMSDLL 220 230 240 250 260 270 280 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|

TcasZ9desA TcasZ9desB DmelZ9des MdomZ9des AdomZ9des HzeaZ9des

QDPILRYQKKYYLFVMPVICFVLPTAAPMYFWGESFKNAFFVN-LFRYCFTLNSTWLVNSAAHLWGSKPY ADPILRFQKKYYTIVMPLVSFVMPTVVPMYLWGETFKNAFLVN-LFRYCLTLNATWLVNSAAHMWGDKPY ADPILMFQKKYYMILMPIACFIIPTVVPMYAWGESFMNAWFVATMFRWCFILNVTWLVNSAAHKFGGRPY ADPIIMFQKKYYLILMPILCFILPTMIPMYGWNESFMNSWFVATMFRWCFILNITWLVNSAAHKFGGKPY QDKIVMFQKKYYLILMPIVCFLIPTTIPVYMWNETWSNAWFVATLFRYTFTLNMTWLVNSAAHMWGSQPY ADPILRFQKKYYLILMPLACFVMPTVIPVYFWGETWTNAFFVAAMFRYAFILNVTWLVNSAAHKWGDKPY 290 300 310 320 330 340 350 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|

TcasZ9desA TcasZ9desB DmelZ9des MdomZ9des AdomZ9des HzeaZ9des

DKFINPAENFAVSVLALGEGWHNFHHTFPWDYKASELGKYSVNFSSAFIDFFAKIGWAYDLKTVSEDLVK DRFINPAENFVVSVLALGEGWHNYHHTFPWDYKTSELGKYSVNFSTAFIDFFAKIGWAYDLKTVSSEMIK DKFINPSENISVAILAFGEGWHNYHHVFPWDYKTAEFGKYSLNFTTAFIDFFAKIGWAYDLKTVSTDIIK DKYINPAENKSVAILAFGEGWHNYHHVFPWDYKTAEFGNYSMNMTTGFIDFFAKIGWAYDLKTVSADIIK DKYINPAENLGVALGAMGEGWHNYHHVFPWDYKAAELGNYRANFTTAFIDFFARIGWAYDLKTVPVSMIQ DKSIKPSENLSVAMFALGEGFHNYHHTFPWDYKTAELGNNKLNFTTTFINFFAKIGWAYDLKTVSDDIVK 360 370 380 390 ....|....|....|....|....|....|....|....|

TcasZ9desA TcasZ9desB DmelZ9des MdomZ9des AdomZ9des HzeaZ9des

KRVLRTGDGSHHVWGWGDMDQALEDYEGAIIKHRKSDDFN KRVTRTGDGTHEIWGWGDKDQSQEDYQDAIITHRKSA--KRVKRTGDGTHATWGWGDVDQPKEEIEDAVITHKKSE--KRVKRTGDGSHATWGWGDKDQPKDEIDNAIIINKKDE--RRVERTGDGSHEVWGWGDKDMPQEDIDGAVIEKRKTQ--NRVKRTGDGSHHLWGWGDENQSKEEIDAAIRINPKDD---

Fig. 2. Amino acid sequence alignment of TcasZ9desA and TcasZ9desB with representatives of other insect Orders: Diptera D. melanogaster (Dmel), M. domestica (Mdom); Orthoptera A. domesticus (Adom); and Lepidoptera H. zea (Δ9 (16 N 18)). Solid shading denotes conservation of aa identity and grey shading for residues with similar characteristics. Conserved histidine boxes are indicated by dashed lines, conserved motifs (as described in Section 3.1) are given by open boxes.

variable without affecting substrate- or regio-specificity of the enzyme; Liu et al. (2004) reported similar activities from two Z9 desaturases which differed only in one sequence having an additional 19 residues in the N-terminus of the protein. Other features of TcasZ9desA and TcasZ9desB include the NPAE signature motif located just prior to the third histidine box (Fig. 2) which is also found in the A. domesticus, and M. domestica Z9 desaturases and may imply a common lineage for these genes (Knipple et al., 2002). Both TcasZ9desA and B are predicted to possess 4 transmembrane regions (data not shown) and these are located in similar positions to those of the well-characterised mouse stearoyl CoA desaturase (Man et al., 2006).

3.2. Functional characterization of TcasZ9desA and B The S. cerevisiae strain ole1 lacks a Z9 desaturase and requires Z9 unsaturated fatty acids for growth. Complementation of the S. cerevisiae ole1 auxotroph can be achieved by the heterologous expression of a Z9 desaturase that acts on C16:0 or C18:0 fatty acids (Stuckey et al., 1990). When pYES-TcasZ9desA and pYES-TcasZ9desB were transformed into S. cerevisiae ole1, recombinants were capable of growing on media without the addition of unsaturated fatty acids suggesting the gene products were likely Z9 desaturases (data not shown). Analysis of FAME extracted from ole1 transformed with pYES-TcasZ9desA and pYES-TcasZ9desB by GC clearly showed

I. Horne et al. / Gene 468 (2010) 41–47

45

Fig. 3. Gas chromatography analysis of fatty acid methyl esters (FAME) from ole1 yeast cells transformed with (A) pYES-TcasZ9desA and (B) pYES-TcasZ9desB and a control, (C) pYES. Peaks corresponding to the production of 9-hexadecanoic acid (16:1) and 9-octadecanoic acid (18:1) are indicated on the chromatogram. The Z9 unsaturated fatty acid 17:1 is required for ole1 growth in the absence of a Z9 desaturase.

production of large amounts of 9-octadecanoic acid and smaller quantities of 9-hexadecanoic acid (Fig. 3A and B) that are absent in ole1 transformed with pYES (Fig. 3C) with the conversion data summarised in Table 3. Chain elongation of 9-hexadecanoic acid formed by pYES-TcasZ9desA and pYES-TcasZ9desB to 11-octadecanoic acid by native yeast elongases was not observed in the GC spectra (Fig. 3A and B). ole1 transformed with empty vector pYES required the Z9 unsaturated fatty acid C17:1 for growth and this appears in the spectrum (Fig. 3C). Formation of Z9 octadecenoic acid by TcasZ9desA and B expressed in yeast was confirmed by GCMS comparison of retention time and mass spectra with authentic standards of oleic and palmitoleic acids and analysis of the mass spectra of the DMOX derivatives of each fatty acid sample (data not shown). Thus both

Table 3 Percentage conversion of saturated fatty acids to their Z9 monounsaturated form by T castaneum Z9 desaturases expressed in Saccharomyces cerevisiae strain ole1. Fatty acid

pYES-TcasZ9desA (%)

pYES-TcasZ9desB (%)

pYES (%)

C14:1Δ9 C16:1Δ9 C18:1Δ9 C20:1Δ9

n.d. 11.8 88.7 n.d.

n.d. 8.9 79.2 n.d.

n.d. n.d. n.d. n.d.

TcasZ9desA and B show a preference for stearic acid over palmitic acid substrates which they convert to the Z9 unsaturated fatty acids. 3.3. Phylogenetic analysis and evolutionary change in TcasZ9desA and B Desaturases involved in pheromone synthesis are generally under intense selection pressure which has resulted in changed specificities for chain length and regio- and stereo-selectivity in the encoded proteins (Roelofs and Rooney, 2003; Knipple et al., 2002) but desaturases involved in homeostasis are much more conserved. We examined the ratio of non-synonymous to synonymous nucleotide changes (dN/dS ratio) between TcasZ9desA and TcasZ9desB as a measure of potential selection pressure. The determined dN/dS ratio was 0.09 suggesting these genes are not under positive selection and are not diverging in sequence consistent with their roles as homeostatic Z9 desaturases. An unrooted Neighbor-joining phylogenetic tree of homeostatic, pheromone- or sex-specific and unknown function insect desaturase aa sequences was constructed with TcasZ9desA (HM234671) and B (HM234672) (Fig. 4). While the two T. castaneum desaturases were separated into a clade in the tree there was only weak statistical support for this arrangement although they are positioned close to the Δ9 C18 N 16 clade of moth sequences with which they share similar functional activities. In contrast to the close relationship of the Δ9 and

46

I. Horne et al. / Gene 468 (2010) 41–47

AF518020 C. parallela

99 93

AF518021 C. parallela

98

AY061988 E. . postvittana AF268275 P. octo

70

AF243047 O. nubilalis

Δ9 (16>18)

AM076338 M. sexta

99

AF272343 H. zea AY362878 S. littoralis

99

EU285579 M. brassicae

63

AGAP001713-PA A.gambiae AJ245747 D. melanogaster 63

Δ9 (16>18)

AB300221 D. antiqua

99

AF417841 M. domestica

81 67

AJ271415 D. melanogaster

Δ9 (14)

AF430246 O. nubilalis 99 99

AF402775 E. postvittana AF518010 C. parallela AF038050 T. ni

95

Δ9 (18>16)

AF272345 H. zea

99 96

AY362877 S. littoralis HM234671 T. castaneum HM234672 T. castaneum

99

AJ271414 D. melanogaster DQ191800 D. abbreviatus AF338465 A. domesticus

53

EU159448 A. domesticus AF518008 C. parallela

Δ9(18>16) Δ12 Δ9 (14-26)

AGAP003418-PA A. gambiae

67

LOC551527 A. mellifera

51

AF518012 C. parallela AY017379 P. octo 99

AF157627 B. mori

71

AF272342 H. zea

91 87

AY362879 S. littoralis

56

EF150363 T. pityocampa

Δ10 – Δ13

AM158251 M. sexta AF441221 O. nubilalis

80

AF518014 C. parallela AY049741 E. postvittana

99

EU159449 T. castaneum

Δ12

AGAP003051-PA A. gambiae LOC552176 A. mellifera

99

AGAP003049-PA A. gambiae AF441220 O. nubilalis

Δ14

AGAP004572-PA A. gambiae

50

0.1

Fig. 4. Neighbor-joining phylogenetic tree of T. castaneum and other desaturase representatives from five insect Orders. Statistical support for nodes with bootstrap values (resampling size of 1500) are given at branch points; those with N50% support are indicated. The insect names use abbreviated genus name and species names together with Genbank nucleic acid sequence accession numbers. Full insect names are given in Section 2.3. Characterised activities of the desaturases (ΔX; where X is the first point of unsaturation in the molecule and numbers in parenthesis refer to the carbon chain length specificity) are shown on the left hand side of the tree.

Δ12 desaturases of the cricket Acheta domesticus in the phylogenetic tree where the two desaturases share a clade, the Δ12 desaturase from T. castaneum is located in a distant branch to that of the Δ9 desaturases. There are two large clusters of moth desaturases that are strongly supported; the well characterised Δ9(16 N 18) and Δ9 (18 N 16) lineages which differ in their preferred substrates (Knipple et al., 2002). The phylogenetic tree also shows that the D. melanogaster Z9 desaturases (AJ271415 and AJ271415) that are sex-specific or involved in pheromone production have diverged from the homeostatic Z9 desaturases. By contrast, several of the moth Z9 desaturases

were identified in pheromone glands (AF272343 and EU285579) but cluster with the homeostatic desaturases of insect fat body.

3.4. Expression of TcasZ9desA and B with developmental stage Expression of the two Z9 desaturases was quantified from transcripts present in T. castaneum life stages and found to follow a similar pattern, that is, pupal expression was lowest for both genes (Fig. 5). There were minor differences in expression level of the two

Scaled expression

I. Horne et al. / Gene 468 (2010) 41–47

1.6

TcasZ9desA

1.4

TcasZ9desB

References

1.2 1.0 0.8 0.6 0.4 0.2 0.0 Adult

47

Pupae Developmental stage

Larvae

Fig. 5. Quantitative expression (± SD) of TcasZ9desA and B in whole adult, pupae and larvae of T. castaneum. Data were normalised to the relative expression of reference genes tubulin and GAPDH whose expression levels were also investigated at each life stages.

genes; TcasZ9desA was more highly expressed in adults but TcasZ9desB was more highly expressed in larvae as shown in Fig. 5. T. castaneum has two closely related Z9 desaturase sequences which show the same substrate and regiospecificity and level of activity when heterologously expressed in yeast but no positive selection could be found acting on the two genes. Multiple Z9 desaturases involved in homeostasis have been previously reported but they are not common. Mice have three isoforms of stearoyl-CoA desaturase that share N75% nucleotide similarity but have different regulatory and tissue specificities (Ntambi and Miyazaki, 2003) and Aspergillus nidulans has two Z9 desaturases where the lack of both genes is lethal. One Z9 desaturase, sdeA, was found to be required for growth and development at all temperatures whereas sdeB was required for low temperature growth (Wilson et al., 2004). Several examples of alternative mRNA splice variants that produce identical coding regions but differ in 5′UTR regions have been described for Z9 desaturases of insects including crickets (Riddervold et al., 2002) and flies (Eigenheer et al., 2002) whereas the moth Choristoneura parallela has two transcripts that give two different ORFs, with one coding for an extra 19 aa in the N-terminus but otherwise the sequences were identical (Liu et al., 2004). Therefore multiple Z9 desaturase genes or alternative transcripts appear to be important for controlled cellular development within tissues of multicellular organisms and we suggest that this is the case for T. castaneum. 4. Conclusion We have identified two conserved Z9 desaturases in T. castaneum both having a strong preference for stearic acid substrate and similar activity when expressed heterologously. Considering their close relationship to other homeostatic insect Z9 desaturases these genes are most likely to be involved in desaturating fatty acids for essential purposes in the beetle. Quantitative analysis of TcasZ9desA and B transcripts showed both genes are similarly expressed in all insect life stages with pupae having the lowest expression level. Hence, expression of two conserved Z9 desaturase genes in T. castaneum may be due to a requirement for tissue-specific expression. These findings also provide a sound basis for further examination of evolutionary and functional comparisons with other desaturases in the organism. Acknowledgments The authors would like to acknowledge the financial support of the Grains Research and Development Corporation (GRDC), and thank Greg Dojchinov for supplying T. castaneum beetles and Drs Matt Taylor, Shoko Okada and Xue-Rong Zhou for their helpful comments on the manuscript.

Altschul, S.F., et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Dallerac, R., Labeur, C., Jallon, J.-M., Knipple, D.C., Roelofs, W.L., Wicker-Thomas, C., 2000. A Δ9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 97, 9449–9454. Dobson, G., Christie, W.W., 2002. Mass spectrometry of fatty acid derivatives. Eur. J. Lipid Sci. Technol. 104, 36–43. Eigenheer, A.L., Young, S., Blomquist, G.J., Borgeson, C.E., Tillman, J.A., Tittiger, C., 2002. Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences. Insect Mol. Biol. 11, 533–542. Grimaldi, D., Engel, M.S., 2005. Evolution of the Insects. Cambridge University Press, New York. (755 pp.). Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J., 2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19. Hofmann, K., Stoffel, W., 1993. TMbase—a database of membrane spanning protein segments. Biol. Chem. Hoppe Seyler 374, 166. Kaiser, C., Michaelis, S., Mitchel, A., 1994. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Manual. . Kayukawa, T., Chen, B., Hoshizaki, S., Ishikawa, Y., 2007. Upregulation of a desaturase is associated with the enhancement of cold hardiness in the onion maggot, Delia antique. Insect Biochem. Mol. Biol. 37, 1160–1167. Knipple, D.C., Rosenfield, C.-L., Nielsen, R., You, K.M., Jeong, S.E., 2002. Evolution of the integral membrane desaturase gene family in moths and flies. Genetics 162, 1737–1752. Labeur, C., Dallerac, R., Wicker-Thomas, C., 2002. Involvement of desat1 gene in control of Drosophila melanogaster pheromone biosynthesis. Genetica 114, 269–274. Liu, W., Rooney, A.P., Xue, B., Roelofs, W.L., 2004. Desaturases from the spotted fireworm moth (Choristoneura parallela) shed light on the evolutionary origins of novel moth sex pheromone desaturases. Gene 342, 303–311. Man, W.C., Miyazaki, M., Chu, K., Ntambi, J.M., 2006. Membrane topology of mouse stearoyl-CoA desaturase 1. J. Biol. Chem. 281, 1251–1260. Ntambi, J.M., Miyazaki, M., 2003. Recent insights into stearoyl-CoA desaturase 1. Curr. Opin. Lipidol. 14, 255–261. Park, H.Y., Kim, M.S., Paek, A., Jeong, S.E., Knipple, D.C., 2008. An abundant acyl-CoA (delta9) desaturase transcript in pheromone glands of the cabbage moth, Mamestra brassicae, encodes a catalytically inactive protein. Insect Biochem. Mol. Biol. 38, 581–595. Riddervold, M.H., Tittiger, C., Blomquist, G.J., Borgeson, C.E., 2002. Biochemical and molecular characterization of house cricket (Acheta domesticus, Orthoptera: Gryllidae) Δ9 desaturase. Insect Biochem. Mol. Biol. 32, 1731–1740. Roelofs, W.L., Rooney, A.P., 2003. Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera. Proc. Natl. Acad. Sci. U.S.A. 100, 9179–9184. Rosenfield, C.-L., You, K.M., Marsella-Herrick, P., Roelofs, W.L., Knipple, D.C., 2001. Structural and functional conservation and divergence among acyl-CoA desaturase-encoding genes of two noctuid species, the corm earworm, Helicoverpa zea, and the cabbage looper, Trichoplusia ni. Insect Biochem. Mol. Biol. 31, 949–964. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 949–964. Shanklin, J., Whittle, E., Fox, B.G., 1994. Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33, 12787–12794. Stuckey, J.E., McDonough, V.M., Martin, C.E., 1990. The OLE1 gene of Saccharomyces cerevisiae encodes the Δ9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J. Biol. Chem. 261, 13230–13235. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Tiku, P.E., Gracey, A.Y., Macartney, A.I., Beynon, R.J., Cossins, A.R., 1996. Cold-induced expression of delta 9-desaturase in carp by transcriptional and posttranslational mechanisms. Science 271, 815–818. Tribolium Genome Sequencing Consortium, 2008. The genome of the model beetle and pest Tribolium castaneum. Nature 452, 949–955. Wilson, R.A., Chang, P.-K., Dobrzyn, A., Ntambi, J.M., Zarnowski, R., Keller, N.P., 2004. Two Δ9-stearic acid desaturases are required for Aspergillus nidulans growth and development. Fungal Genet. Biol. 41, 501–509. Xue, B., Rooney, A.P., Kajikawa, M., Okada, N., Roelofs, W.L., 2007. Novel sex pheromone desaturases in the genomes of corn borers generated through gene duplication and retroposon fusion. Proc. Natl. Acad. Sci. U.S.A. 104, 4467–4472. Yu, Q.T., Liu, B.N., Zhang, J.Y., Huang, Z.H., 1988. Location of methyl branchings in fatty acids: fatty acids in uropygial secretion of Shanghai duck by GC-MS of 4, 4dimethyloxazoline derivatives. Lipids 23, 804–810. Zhou, Z.R., Horne, I., Damcevski, K., Haritos, V., Green, A., Singh, S., 2008. Isolation and functional characterization of two independently-evolved acyl-CoA Δ12-desaturase genes from insects. Insect Mol. Biol. 17, 667–676. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.