Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

Identification of WP5212-like DNA sequences from the hexaploid wheat genome

A total of 25 positive BAC clones were identified by screening 24 high-density filters of the hexaploid wheat Triticum aestivum cultivar Glenlea BAC library (3.1× haploid genome coverage) with the WP5212 specific probe. Twenty-two positive clones were confirmed to contain WP5212-like inserts through PCR analysis. Secondary screening by DNA sequencing confirmed three unique sequences.

Sequencing of WP5212-like sequences from positive BAC clones

Out of 22 candidates, three unique clones, 1333A17, 895N14, and 1324M8 were chosen to be sequenced. Sequencing primers were designed based on conserved regions in cDNAs from WP5212, barley Beg1 [GenBank: M64372] and maize Glb1 [GenBank: M24845] determined by sequence alignment. Sequencing was conducted by primer walking. Obtained DNA sequences were assembled into contigs of 4406, 3671 and 1457 bp nucleotides for 1333A17, 895N14 and 1324M8, respectively.

Sequence characterization and open reading frame identification

Contigs from clones 1333A17 and 895N14 contained full genomic sequences for a globulin gene as determined by open reading frame analysis. The globulin gene open reading frame starts at base pair 1213 in BAC clone 1333A17 and at base pair 309 in BAC clone 895N14. For the 1324M8 clone, 1457 bp ORF was delineated, but no stop codon was identified. However, a partial 5' coding sequence was identified, starting at position 109 in 1324M8 contig.

No similarity to previously identified wheat globulin genes was found, thus the specific genes were named Glo-3A, Glo-3B and Glo-3C in the order they were identified (originating from BAC clones 1333A17, 895N14, and 1324M8, respectively). All three sequences have been entered into GenBank [GenBank: FJ439135, FJ439136 and FJ439134]. Putative open reading frames, the transcription start sites and the polyadenylation signal sequences were identified (Figure 1A). All sequences contain five to seven exons and four to six introns.

Comparisons between the cDNA clone WP5212 and the coding region of Glo-3A showed 99% identity at the nucleotide level. Translated sequence alignment with the predicted amino acid sequence of cDNA clone WP5212 resulted in 99% identity: 583 identical, 3 conserved (G5A, A7V, R102H) and 2 non-conserved (R43Q, A549T) amino acids out of 588 total amino acid residues (Figure 2). Similarly, 99% identity was shared between Glo-3A and the assembled wheat transcript [TIGR: TA61374_4565], composed of 230 T. aestivum ESTs, known to be 100% identical to WP5212. Importantly, taken together these results demonstrate that the correct terminology for the WP5212 homologue in wheat (previously called Glb1 homologue) should now be named Glo-3A.

The alignment of Glo-3B with Glo-3C indicated that both genes share 88% and 70% identity at the nucleotide and amino acid level, respectively. Glo-3B and Glo-3C share 91% and 95% identity with the Glo-3A sequence at the nucleotide level, and 74% and 93% identity at the amino acid level. The deduced translation product identified two cupin domains in Glo-3A (at 136–260 and 341–508) and one in Glo-3B (299–425) and Glo-3C (150–264). Cupin domains are common features among vicilins. They are important for nutrition and also play a role in immunoactivation [22, 23]. Proteins expressed from Glo-3A and Glo-3B would be 66.3 kDa and 56.9 kDa, and have predicted pIs of 7.7 and 7.5, respectively.

Promoter identification and regulatory elements

The 1200 bp of the 5' flanking sequence of Glo-3A were analyzed to identify a potential promoter using TSSP, a plant promoter recognition program http://www.softberry.com and PlantProm database [24]. A putative promoter region was indicated to be between -897 and -43 upstream of the ATG start codon. The promoter region was analyzed for potential cis-acting elements using the PLACE database [25]. Multiple putative cis-acting elements were identified (Figure 1B and see Additional file 1). The promoter region includes potential binding sites for transcription factors, such as bZIP and DOF. Presence of the abscisic acid response element ABRE suggests the expression of Glo-3A is hormone-regulated. Several elements related to tissue specific expression were also found, including E-box, RY repeat, Pyrimidine box, C-box, T-box, and a Prolamin-box.

Transcriptional activity

The presence of Glo-3A mRNA in T. aestivum cv. Glenlea seeds 16 days post anthesis demonstrates that this gene is actively transcribed. Primers that correspond to highly conserved regions of Glo-3A, Glo-3B, Glo-3C, barley Beg1 and maize Glb1, so they could bind to a transcript from any of the three sequences, were used to identify Glo-3 transcripts. Since the 3' end of Glo-3C was not recovered during primer walking, we designed our primers on the assumption that the 3' region of Glo-3C was similar to the other Glo-3 genes and, if transcribed, would be amplified during RT-PCR analysis. A cDNA fragment of 900 bp, sequenced from the RT-PCR product, was 100% identical with the Glo-3A predicted cDNA sequence in the studied region and corroborated the predicted intron-exon structures (Data not shown). However, RT-PCR products for Glo-3B and Glo-3C were not observed. Transcriptional activity of Glo-3A was further supported by BLAST analysis of wheat EST databases. At least 729 EST sequences from the hexaploid wheat T. aestivum shared high similarity with Glo-3A and Glo-3B and 250 ESTs with Glo-3C (see Additional file 2). Among the total of 740 ESTs matching to Glo-3A, 722 ESTs were from the developing or mature seed. Relatively few, only 25, ESTs were from T. aestivum Glenlea developing seed library 15 DPA, the same cultivar and sampling time used for constructing the genomic library screened in this study and RT-PCR analysis, respectively. With respect to tissue-specific Glo-3 gene expression, eight EST sequences from T. turgidum durum seedling library were identified. Also, a highly homologous EST clone [GenBank: BQ802077] from T. monococcum EST vernalized apex library was found. Mapped ESTs (from Chinese Spring deletion lines) indicated the location of Glo-3A to be on the 4AL and/or 4BS chromosome [GrainGenes: BE590748].

Gene Family Size: determining Glo-3gene copy number in wheat

Wheat cultivars were screened by Southern blot to identify possible genetic lines that might be devoid of WP5212-like proteins. DNA was extracted from cultivars representing all ploidy levels: T. aestivum AC Barrie (AABBDD genome), T. aestivum Glenlea (AABBDD genome), T. aestivum Chinese Spring (AABBDD genome), T. aestivum Spelta (AABBDD genome), T. turgidum durum Kyle (AABB genome), T. turgidum dicoccum (AABB genome), T. monococcum monococcum (AA genome), Aegilops speltoides (BB genome), and Ae. tauschii (DD genome). A 650 bp fragment, amplified from the highly conserved region among all of the three BAC clones was used as a hybridization probe. All of the cultivars examined contained Glo-3 genes. Based solely on the number of bands, there are at least two copies of Glo-3 genes in A, B and D diploid genomes and at least four homologous copies in tetraploid and hexaploid genomes (Figure 3).

Immunolocalization

Immunofluorescence labeling using rabbit anti-WP5212 antibodies revealed the localization of corresponding globulin protein in the aleurone layer and embryo, but not the endosperm of wheat seed sections (Figure 4A, C). Observed fluorescence in wheat seed coat is attributable to background non-specific binding. WP5212 was shown to share immunodominant epitopes to the peanut allergen Ara h1. The same antibodies bound proteins from peanut cotyledon (Figure 4C). WP5212 pre-immune serum did not localize to the embryo, endosperm or aleurone layer in wheat or cotyledons in peanut (Figure 4B, D, F).

Plant material and preparation of high molecular weight DNA

Wheat Triticum aestivum cv. AC Barrie seeds were provided by Agriculture and Agri-Food Canada, Indian Head Research Farm and Seed Increase Unit (Indian Head, SK). T. aestivum cv. Glenlea, T. aestivum cv. Chinese Spring, T. aestivum cv. Spelta (CDC Bavaria), T. turgidum durum cv. Kyle (CN42944), T. turgidum diccoccum (CItr 3686), T. monococcum monococcum, Aegilops speltoides (PI 542261), and Ae. tauschii (WGRC2375) seeds were obtained from USDA National Small Grains Research Facility (Idaho). Seeds were grown under standard greenhouse conditions, until shoots were 15–20 cm long. Large-scale purification of DNA was performed as described previously [41]. Briefly, leaves were cut and ground immediately in liquid nitrogen. Thirty mL of 65°C extraction buffer (100 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), and 500 mM NaCl) was added to 20 mL of frozen tissue in 50 mL falcon tubes and the contents were shaken. Two millilitres of 20% sodium dodecyl sulphate (SDS) was added to each tube and the tubes were shaken vigorously. The samples were incubated at 65°C for 10 min. Ten mL of 5 M potassium acetate was added to each tube, the tubes were shaken vigorously and placed on ice for 20 min. Samples were clarified by centrifugation at 9000 rpm for 20 min at 4°C in a Beckman JA-12 rotor (Beckman Coulter, Fullerton, California) followed by filtration of the supernatants through one layer of sterile Medicom gauze into sterile 50 mL tubes. RNAse A (10 mg/mL stock) was added at a concentration of 2 μg/mL sample. After ethanol precipitation, the DNA was removed from each tube using a glass Pasteur pipette. The DNA was washed once in 70% ethanol, dried, and dissolved in TE buffer (10 mM Tris-HCl (pH 8.0) – 1 mM EDTA (pH 8.0)) at a concentration of approximately 5–10 mg/mL.

Restriction digests and Southern blotting

DNA samples were digested with restriction enzymes, Xba I and Xho I in buffer supplied by the manufacturer (Invitrogen). Gels were alkaline blotted onto Hybond N+ (Amersham, Piscataway, N.J.) membranes using a standard capillary transfer setup with glass plates for support.

Hybridization studies – BAC library screen and Southern blots

The Glenlea BAC library, kindly supplied by Dr. Cloutier from AAFC-Winnipeg, contains 656,640 clones with an estimated 3.1× haploid genome coverage and has been gridded onto 24 high-density filters [42]. A 459 bp probe (amplified using primers 5'AAAAAGCAGGCTTTCGACGAAGTGTCCAGG 3'and 5'AGAAAGCTGGGTTGCCCAAGAGACTACCCA 3') for the BAC library screening and 650 bp probe (amplified using primers Glb09F and Glb09R) for the Southern blot, consisting of the partial coding regions of WP5212 cDNA clone [11] was labeled with [α-³²P]dCTP using Ready-to-Go DNA labeling beads (Amersham, Baie d'Urfé, QC) following the manufacturer's instructions and used to screen the filters and membranes. Hybridization was performed as described by Nilmalgoda and co-workers [42]. In short, hybridization buffer was prepared as described by Church and Gilbert (1984), but without BSA. The blots were pre-hybridized overnight at 65°C. The probe was denatured by 10 min boiling, and added to the hybridization tubes. Hybridization was carried out overnight at 65°C. Filters were washed at 65°C in increasingly stringent buffers (2 × sodium chloride/sodium citrate SSC, 0.1% SDS to 0.1× SSC, 0.1% SDS) until counts were approximately 1000 cpm. The following PCR conditions were used for probe preparation: 1× PCR buffer (Invitrogen), 1.5 mmol MgCl₂/L (Invitrogen), 0.2 mmol/L each of the four dNTPs (Invitrogen), 15 pmol each forward and reverse primers, 1.2 U Taq polymerase. The amplification reaction: 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min before cooling to 4°C. The PCR products were purified using the QIAEX II gel extraction kit (Qiagen, Mississauga, ON) according to the manufacturer's instructions.

RT-PCR

RT-PCR was performed following Invitrogen's protocol. In short, total RNA was isolated from ground seeds (100 mg aliquots) of T. aestivum cv. Glenlea, collected 16 days post anthesis. RNA was extracted and purified using RNeasy plant mini kit (Qiagen, Mississauga, ON) according to the manufacturer's instructions. The first strand cDNA was synthesized using the First-Strand Synthesis System for RT-PCR from Invitrogen according to the manufacturer's protocol. The RT-PCR product was amplified with WPF1 and WPR1 primer pair (design based on three Glo-3 sequences [GenBank: FJ439134-FJ439136], barley Beg1 [GenBank: M64372] and maize Glb1 [GenBank: M24845] (see Additional file 3). Expected RT-PCR product sizes on mRNA were 1177 bp and 949 bp for Glo-3A and Glo-3B, respectively. The same primer pair would amplify PCR products of 1562 bp for Glo-3A and 1604 bp of Glo-3B on a DNA template. RT-PCR products were cloned into a plasmid vector pGEM-T Easy (Promega, Madison, WI), and their nucleotide sequences were determined using WPR1, WPR2, WPF1, Glb1R, Glb02F and Glb08F primers (see Additional file 3).

Sequencing

All sequencing was performed at the Ottawa Hospital Research Institute, using Applied Biosystems 3730 DNA Analyzer. Primers used for sequencing are shown in Additional file 3. The sequences identified in this paper have been deposited to GenBank [GenBank: FJ439134; FJ439135; FJ439136].

Sequence analysis

The contig sequences were analyzed using the NCBI ORF finder http://www.ncbi.nih.gov/gorf/gorf.html and FGENESH 3.0 alpha http://www.softberry.com to determine the predicted protein sequence. The conceptually translated proteins were aligned using ClustalX and BioEdit programs [43]. Regulatory elements from the promoter region were identified using the PLACE database http://www.dna.affrc.go.jp/PLACE/[25]. BLASTn analyses were performed using NCBI databases [44], Plant Transcript Assembly database http://blast.jcvi.org/euk-blast/plantta_blast.cgi, and wheat mapped EST database (http://wheat.pw.usda.gov/GG2/blast.shtml; all URLs last viewed 22.03.09). EST abundance for each Glo-3 mRNA was measured using MegaBlast against GenBank "non-mouse and non-human EST database limited to Triticum (total of 1,106,281 sequences, 22.03.09). EST sequences with minimum identity of 86% and minimum E value of 1e-07 were chosen. Presence of the conserved domains was screened with NCBI CDD database http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml[45].

WP5212 polyclonal serum production

Antigenic WP5212 peptides were predicted by Sigma-Genosys (The Woodlands, TX) technical services (proprietary software). Two WP5212 specific peptides were synthesized for the purpose of polyclonal WP5212 antisera production (Sigma-Genosys). Peptide 1, a 77% pure 16 residue peptide (CRDTFNLLEQRPKIAN), was conjugated to the carrier protein Keyhole-limpet hemocyanin for immunization. Peptide 2 was a 51% pure 15 residue peptide (RGDEAVEAFLRMATA). The purity was determined by mass spectral and HPLC analyses performed by Sigma-Genosys.

The polyclonal antibody production was performed by Sigma-Genosys. Pre-immune serum was collected from two rabbits, after which they were each co-immunized with 200 μg of peptides 1 and 2 in Complete Freund's Adjuvant (day 0). The rabbits were co-immunized with 100 μg of peptides 1 and 2 in Incomplete Freund's Adjuvant on days 14, 28, 42, 56 and 70. Production bleeds from both rabbits were collected on day 40, 63 and 77. WP5212-specific polyclonal antibody production was assessed by 1D SDS-PAGE and Western blotting of recombinant WP5212 isolated from baculovirus infected insect cell lysate (data not shown). Production bleeds were compared to pre-immune serum samples to confirm antibody production.

LR White sectioning

All fixation and embedding of seeds was performed at 4°C. For London Resin White (LR White) sectioning: mature seeds were fixed in a 4% paraformaldehyde, 0.1 M potassium phosphate buffer (pH 7) for two days. Seeds were placed in two changes of 0.1 M phosphate buffer pH 7 for two hours each, and cut with a razor blade into very thin (2 mm) slices that were placed directly in 70% (v/v) ethanol for one day. Seeds were placed sequentially in 85% ethanol, 100% ethanol and a mixture of 20, 40, 60, 80% (v/v) ethanol: unpolymerized Medium LR White (Sigma-Aldrich, Oakville, ON) for a minimum of two hours per change. Seeds were moved to 100% unpolymerized LR White, and three changes of resin were performed, with a minimum of 12 hours between each change. Finally, seeds were moved to 0.5 ml Beem (Fisher Scientific, Ottawa, ON) capsules containing unpolymerized LR White, and cast at 60°C in a dry oven for four days to polymerize the resin. Polymerized LR White containing seeds were mounted and sectioned with a LKB Ultramicrotome. 2 μm thick sections were placed on Fisher Superfrost Plus slides (Fisher Scientific, Ottawa, ON), and heated on a hot plate for one minute to adhere the sections to the slide.

Immunofluorescent staining

Mounted sections of wheat AC Barrie and peanut Valencia seeds were washed three times for 10 minutes each in PBS. For primary and secondary immunostaining, the slides were washed three times for 10 minutes each in PBS. Sections were incubated with the primary antibody for one hour at room temperature in a humidity chamber. A 1:1000 (wheat embryo and peanut cotyledon) or 1:5000 (wheat aleurone and endosperm) dilution of the WP5212 antibody was incubated with the sections. After the primary antibody incubation, slides were rinsed three times for 10 minutes each in PBS. 20 μl of the 1:400 dilution of anti-rabbit conjugated Alexa Fluor antibody in PBS was pipetted onto each slide and incubated at room temperature for one hour. Slides were rinsed three times for 10 minutes each in PBS and dried. A drop of Prolong^® Gold antifade reagent (Invitrogen, Burlington, ON) was added to each section, 0.2 mm cover slips (Fisher Scientific, Ottawa, ON) were overlayed, and slides were dried overnight at room temperature in the dark. Slides were stored at -20°C. Fluorescent and phase contrast images were visualized using a Zeiss Axiophot microscope (Oberkochen, Germany) equipped with an Olympus DP70 CCD camera. Imaging software, MetaMorph (MetaMorph Imaging Systems, Sunnyvale, California), was used to capture images.