Molecular characterization and expression of α-gliadin genes from wheat cultivar Dacke in Bg 250 rice variety

ABSTRACT

The main seed storage protein in wheat is gluten. It consists of gliadin and glutenins. Gluten gives high elasticity and extensibility during bread making, facilitating the formation of the dough. Rice is the staple food of Sri Lankans but, it has poor dough making ability compared to wheat. The aim of the present work was to characterize, clone and express α-gliadin in the T₀ generation of Bg 250 rice variety as a preliminary step in improving the dough making ability of rice flour. Five α-gliadin recombinant pCR™2.1-TOPO® clones were selected for sequence analysis. Of the five clones, two functional genes and three pseudogenes were identified. Phylogenetic analysis revealed the two functional genes, (accession numbers KC660359 and KC660358) to be closely related to the α-gliadin genes of Triticum monococcum. The α-gliadin gene (KC660359) contained five cysteine residues, one less than the normal occurrence of cysteine residues in α-gliadin genes. To date there are no reports on expression of gliadin gene in transgenic rice. This novel gene was successfully expressed in the Sri Lankan rice variety Bg 250 under the control of the rice GluB-1 endosperm specific promoter.

INTRODUCTION

Wheat flour is unique as its dough exhibits the rheological properties required to produce leavened bread. The quality of the flour is determined by the chemical and physical properties of the gluten. Gluten consists of two groups of proteins: gliadin and glutenins. Gliadin accounts for 40–50% of the total seed storage proteins of wheat.¹ It is a heterogeneous mixture of a monomeric protein of 30–78 kDa, soluble in 70% alcohol. Based on the electrophoretic mobility in acid–PAGE, gliadins have been divided into three groups: α/β-, γ-, and ω-.^2,3 The ω- gliadins differ in their amino acid composition from the other two groups and they lack cysteine residues. The α-gliadins are the most abundant, accounting for 15–30% of wheat seed proteins. Hence, they are the most consumed storage proteins by humans.^4–6

The α-gliadin genes are encoded by a multigene family^7–9 at the Gli-2 loci on the short arms of the group 6 chromosomes.¹⁰ In the analysis of the gliadin gene copy number, a major discrepancy has been observed between Southern blot analysis and 2D PAGE analysis.^4,11,12 The possible causes for discrepancies have been attributed to frequent imperfect pairing and recombination, or unequal crossover of the ancestral genes within the families resulting in pseudogenes.^7-9 Furthermore, almost 50% of the α-gliadin genes are inactive due to base substitution of glutamine (CAA) to a stop codon (TAA) irrespective of its location in the gliadin gene.¹²

The primary structure of α-gliadin is depicted in Fig. 1. It comprises of a short signal peptide (20 amino acids) and 5 distinct domains: a repetitive domain, a polyglutamine domain I, a unique domain I, a polyglutamine domain II, and a unique domain II.¹² Six conserved cysteine residues of α-gliadins are in the unique domains I & II and form three intramolecular disulfide bonds.¹³ However, Kasarda (1989)¹⁴ proposed that gliadins with an odd number of cysteines could form intermolecular disulfide bonds with Low Molecular weight (LMW) and High Molecular Weight (HMW) glutenin and thus participate in gluten polymer formation. Different lengths of α-gliadin protein have been identified. This is due to the different number of glutamine codons in the two polyglutamine domains of the gene.⁵

FIGURE 1.

Model of α-gliadin polypeptide based on Anderson et al. (1997)¹² and Muller and Wieser (1995).¹³

The gliadin/glutenin ratio is important for the preparation of quality bread because they play different roles in the gluten network. The elasticity and the strength of the dough is provided by glutenin while gliadins provide plasticity to the glutenin polymeric network.¹⁵ In the past years, glutenin genes (LMW &HMW) have been successfully expressed in E. coli¹⁶ as well as transferred to different wheat^17-20 and rice cultivars.^21–26 Gliadin has been expressed in E. coli.²⁷ However, reports on the transgenic expression of gliadin in plants are limited. High-level in vitro expression of gliadin genes is still difficult, and the genetic and biochemical mechanisms remain unknown.²⁷ There are no reports on the expression of gliadin protein in the rice endosperm.

The aim of this study was to transform the Sri Lankan rice cultivar Bg 250 with the α-gliadin gene as a preliminary step to improve the dough making characteristics of rice flour.

MATERIALS AND METHODS

Plant Material

Seeds of Bg 250 rice were kindly provided by the Regional Rice Research and Development Centre, Bombuwala, Sri Lanka and the wheat cultivar Dacke variety seeds were kindly provided by the late Professor S.R. Srimanne, Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo, Sri Lanka.

Genomic DNA Extraction and Gene Cloning

Wheat and rice seeds were grown in pots containing soil from a paddy field. DNA was extracted from wheat and rice leaves according to the method described by Pervaiz et al. (2011).²⁸ Primers for PCR amplification of the α-gliadin gene were designed by aligning open reading frame (ORF) sequences of α-gliadin genes of different wheat cultivars in GenBank using clustal W (http://www.ebi.ac.uk/Tools/msa/clustalo/). The two primer sequences are as follows: GFP:5′-GAATTCCCACCATGAAGACCTTTCTCAT-3′ and GRP:5′-GGTAACCCTTCTCAGTTAGTACCGAAGAT−3′ containing EcoRI and BstEII restriction sites (underlined). Each PCR reaction (25 µL) contained 300 ng template DNA, 0.2 mM of each dNTPs, 2.5 mM MgCl₂, 0.2 µM of each of the two primers, 1 X PCR buffer, and 1U Go Taq DNA polymerase (Promega, USA). The following thermal cycler conditions were used: initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 sec and 72 °C for 40 s, and the final extension was 5 min at 72 °C.

Glutelin B1 (GluB-1) promoter primers were designed based on the Oryza sativa japonica coding sequence (Accession number AY427569). The two primer sequences are: GluBF1:5′-GGATCCACAGATTCTTGCTACCAA-3′ and GluBCR:5′-GAATTCAGCTATTTGTACTTGCTTATGGAAACTTAACCT-3′ containing BamHI and EcoRI restriction sites (underlined). These primers were used to amplify the 2300 bp GluB-1 promoter of Oryza sativa indica from the isolated rice genomic DNA. The PCR conditions were as above, and the following thermal cycler conditions were used: Initial denaturation at 94 °C for 5 min and 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 2 min and 30 s followed by a final extension at 72 °C for 15 min.

PCR products were separated on a 0.8% agarose gel and the amplified fragments (gliadin and GluB-1) were purified (Wizard SV gel and the PCR clean – up system kit). The purified PCR fragments were cloned into pCR™2.1-TOPO® vector (Invitrogen TOPO T/A cloning kit) and transformed into E. coli (JM109) competent cells according to the standard protocol provided (Promega, USA). Five clones (pgli 62, 67, 70, 75 & 79) were randomly selected for sequencing. Clones pgli 70, 79 and GluB-1 promoter were completely sequenced (Macrogen Inc, Korea) using vector specific primers while clones 62, 67 and 75 were partially sequenced using gene specific primers (GFP & GRP).

Sequence Data Analysis

The sequence alignments of pgli 62, 67, 70, 75 & 79 gliadin genes were carried out using Clustal W and BioEdit 7.0 (www.mbio.ncsu.edu/BioEdit/bioedit.html) software. Chromosomal location of cloned α-gliadin genes were assigned based on the method of Van Herpen et al. (2006)⁵ and a neighbor-joining phylogenetic tree was constructed for the nucleotide sequences using Mega 7 software based on the distance method (Tamura-Neimodel) with 1000 bootstrap replications. The toxic epitopes in the gliadins were identified according to Cornell and Wills-Johnson (2001).²⁹

Binary Vector Construct and Transformation

The gliadin gene (pgli 79) was digested with restriction enzymes EcoRI and BstEII to clone into the binary vector pCAMBIA 1391Z. The GluB-1 promoter was also cleaved by EcoRI and BamHI to clone into the 5ʹ upstream region of the gliadin gene (Fig. 2). The vector construct was transformed into Agrobacterium GV3101 by the freeze thaw method.³⁰ The recombinant vector was transformed into rice Bg 250 variety as described below. Rice calli were generated in callus induction medium (Table 1). The surface sterilized seeds were cultured on a callus induction medium and incubated at 28 °C for 21 days under dark conditions. Well grown calli were immersed in Agrobacterium tumefacience culture pellet suspended in liquid re-suspension medium (Table 1). The infected calli were cultured on a co–cultivation medium (Table 1). After incubation, the calli were washed with sterile distilled water followed by washing with a series of aqueous solutions containing 1 g/L, 750 mg/L and 500 mg/L cefotaxime. Thereafter, the calli were transferred to a callus induction medium supplemented with cefotaxime (500 mg/mL). After two weeks the calli were transferred to a callus selection medium (Table 1). Healthy portions of the calli were sub cultured into a fresh selection medium twice, at 3–4 week intervals. After three rounds of selection, actively growing calli were transferred to a callus induction medium with cefotaxime and kept in the dark for 2 weeks. The culture plates were then transferred to light and allowed to grow. The green coloured calli were selected and transferred into shoot generation medium (Table 1). Once the shoots proliferated, they were transferred into root generation medium (Table 1). Plantlets with well-developed root systems were transferred to pots containing sterilized soil. Rice panicles appeared after two months and seeds were harvested towards the end of the 4^th month.

FIGURE 2.

Binary vector pCAMBIA 1391Z construct of the gliadin gene under the control of GluB-1 promoter. RB - Right Border; NOS – NOS terminator; GluB-1 - Glutelin B1 promoter; Gliadin - gliadin gene; Hygromycin - hygromycin phosphotransferase gene marker; CAM35S - Cauliflower mosaic virus 35S promoter; LB - Left Border.

TABLE 1.

Media for callus induction and regeneration.

Type of media	Media composition
Callus induction	MS medium, 300 mg/L casein hydroxylate, 300 mg/L proline, 300 mg/L glutamine, 10 g/L ascorbic acid, 3% sucrose, 2 mg/mL 2,4-D, 1 mg/mL BAP, 1 mg/mL NAA and 0.8% agar pH 5.8
Callus re-suspension	MS medium, 300 mg/L casein hydroxylate, 300 mg/L proline, 300 mg/L glutamine and 10 g/L ascorbic acid, 20% sucrose, 10% glucose and 100 μM acetosyringone pH 5.6
Callus co-cultivation	MS medium, 300 mg/L casein hydroxylate, 300 mg/L proline, 300 mg/L glutamine, 10 g/L ascorbic acid, 3% maltose, 10% glucose, 100 μM acetosyringone and 0.3% phytagel pH 5.6
Selection medium	MS medium, 300 mg/L casein hydroxylate, 300 mg/L proline, 300 mg/L glutamine, 10 g/L ascorbic acid, 3% sucrose, 2 mg/mL 2,4-D, 1 mg/mL BAP, 1 mg/mL NAA, 50 mg/mL hygromycin and 0.4% phytagel pH 5.8
Shoot generation	MS medium, 300 mg/L casein hydroxylate, 300 mg/L proline, 300 mg/L glutamine and 10 g/L ascorbic acid, 3 mg/mL BAP, 1.5 mg/mL NAA, 3% maltose, 0.2 g/L Ascorbic acid, 0.1 g/L Sorbitol, 0.1 g/L adenine sulphate, 0.1 g/L cysteine and 0.4% phytagel pH 5.8
Root generation	MS medium and 0.4% phytagel pH 5.8

Confirmation of Transgenes

DNA was extracted from leaves of putative transgenic plants. PCR was performed with GFP & GRP primers as described above to amplify the 861 bp gliadin gene.

SDS–PAGE and Western Blot Analysis

Transgenic rice flour, non-transgenic rice flour and wheat flour (100 mg each) were obtained from seeds for extraction of gliadin as described by Osborne (1907).³¹ The albumin fraction and the globulin fraction were extracted and discarded. The pellet remaining after the globulin extraction was vortexed with de-ionized water (400 µL) for 1 min, then the mixture was centrifuged for 5 min at 2000 g and the supernatant was discarded. It was then extracted with 70% ethanol and the supernatant was freeze dried for SDS-PAGE and western blot analysis.³² Gluten (Sigma, USA) powder (20 mg) and wheat flour were used as positive controls. Primary rabbit polyclonal antibodies were developed by GenScript (USA) against the sequence (SFRPSQQNPQDQGS) of the functional α-gliadin for identification of α-gliadin.

RESULTS

Molecular Characterization of the α-Gliadin Genes

Five clones were selected randomly for the study (Table 2). NCBI BLAST searches of the nucleotide sequences of the 5 clones revealed 4 sequences (pgli 65, 70, 75 & 79) to have a high degree (99%) of identity with typical α-gliadin sequences in GenBank. The clone pgli 62 had 100% similarity to the Chinese Spring wheat cultivar gliadin sequence (GenBank ID AB982269). The accession numbers assigned for the 5 sequences submitted to GenBank were KC660357 (pgli 67), KC660356 (pgli 62), KC660359 (pgli 70), KC245098 (pgli 75) and KC660358 (pgli 79). Amino acid sequences of these genes were aligned for further analysis (Table 2).

TABLE 2.

Cloned gliadin genes.

							Major T cell peptides
Clone name	Accession number	Internal stop codon	No of nucleotides	No of amino acids	No of Cysteine residues	% of Glutamine	glia-α	glia-α2	glia-α9	glia-α20
Pgli 62	KC660356	2	590 bp	197	4	36.55	0	0	1	1
Pgli 67	KC660357	3	403 bp	135	2	20.74	0	0	1	1
Pgli 70	KC660359	0	852 bp	283	6	30.39	0	0	1	1
Pgli 75	KC245098	3	836 bp	278	10	1.79	0	0	1	1
Pgli 79	KC660358	0	840bp	279	5	30.47	0	0	1	1

In silico sequence comparison of the complete (pgli 70 & 79) and partial sequences (pgli 62, 65 & 75) demonstrated distinctive structural features of previously cloned and characterized α-gliadin genes as described below. The functional gliadin gene contained a single exon without introns, conserved N and C terminal domains, a short signal peptide followed by a short N terminal repetitive domain, a poly glutamine domain, a unique domain, a second polyglutamine domain and a second unique domain. In addition to the two full-ORF genes, three pseudogenes containing at least one in-frame stop codon resulting from a base transition (pgli 62) or frame shift mutations (pgli 67 & 75) were identified. The stop codons (TAA or TAG) were formed by single-base C to T substitution, of glutamine (CAA or CAG). The DNA sequences so obtained were translated into protein sequences using BioEdit 7.0 (Fig. 3). The clones pgli 70 & 79 were functional genes coding for proteins of 283 & 279 amino acid residues respectively. The estimated molecular weights of the mature pgli 70 & 79 were approximately 30 kDa. Variations in six amino acid residues were observed among the cloned functional gliadin genes and pgli 70 contains an insertion of 4 amino acids (RQQQ) in the polyglutamine domain I. The N-terminal repetitive region of α-gliadin genes is composed of repeat motifs and was rich in proline and glutamine. The first five amino acid residues (VRVPV) considered as a characteristic sequence of the α-gliadin N- terminal domain² were present in most of the analyzed α-gliadin genes. Six conserved cysteine residues were found in one functional gliadin gene (pgli 70) and in the other (pgli 79) the cysteine residue in the unique domain I, was substituted by arginine. Four celiac disease (CD) toxic epitopes: glia-α2 (PQPQLPYPQ), glia-α9 (PFPQPQLPY), glia-α20 (FRPQQPYPQ), and glia-α (QGSFQPSQQ) have been reported by Van Herpen et al. (2006).⁵ The deduced protein sequences of both pgli 70 & pgli 79 genes contained only glia-α9 and glia-α20 (Fig. 3). Based on these facts, the two genes could be assigned to chromosome 6A.⁵

FIGURE 3.

Multiple alignment of amino acid sequences of the cloned gliadin genes (functional and pseudogenes). X: internal stop codon, C: cysteine residues, R: arginine residues, Black arrows: the positions of signal peptide, N - terminal repetitive domain, polyglutamine domain I, unique domain I, poly glutamine domain II and unique domain II.

Prediction of the Secondary Structure of α-gliadins

The secondary structures of the mature protein of pgli 79 was predicted with the latest online version (3.3) of the PSIPRED server Fig. 4. According to the classification of Li et al. (2014),²⁷ the secondary structure of pgli 79 was classified as type II.

FIGURE 4.

Diagram depicting the secondary structure of pgli 79 gliadin. The probable positions of the α-helices are indicated by a purple cylinder.

Phylogenetic Analysis of α-gliadin Genes

A phylogenetic tree was constructed based on the nucleotide sequences with the signal peptide derived from the 2 cloned functional genes (pgli 70 & 79), together with 12 nucleotide sequences derived from four diploid wheat species (three sequences each from T. monococcum, T.urartu, Ae. speltoides and Ae. tauschii) and LMW glutenin gene of Triticum aestivum (as outer group) extracted from GenBank (Fig. 5). It revealed the nucleotide sequences of functional gliadin (pgli 70 & 79) in this study to be closely related to T. monococcum. This finding is also consistent with the T cell-stimulatory epitopes of gli-α9 & α20 found in the A genome species.⁵

FIGURE 5.

The phylogenic tree based on the nucleotide sequences of the 2 functional α-gliadin genes (with signal peptides) of this study, 12 α-gliadin genes from diploid wheat species and LMW glutenin gene from wheat.

Generation of Transgenic Rice Lines Expressing Pgli 79 α-gliadin Gene

To express gliadin in rice, a binary vector carrying the pgli 79 gene under the control of the rice endosperm-specific glutelin B1 promoter (GluB-1) and the selectable marker gene hygromycin phosphotransferase (HPT) driven by the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 2) was introduced into the rice Bg 250 variety via Agrobacterium-mediated transformation. Figure 6 shows the regeneration of transgenic plants.

FIGURE 6.

Regeneration of Bg 250 rice with transgenes (a) 21 Days old scutellum derived calli, (b) callus proliferation, (c) 4 weeks old transformed callus of indica rice cultivar Bg 250 on selection medium, (d) 12 week old transformed calli of indica rice cultivar Bg 250 on regeneration medium containing hygromycin and cefotaxime, (e) hygromycin resistant proliferating calli on regeneration medium containing cefotaxime, (f) hygromycin resistant proliferating calli on shoot regeneration medium, (g) plantlets in soil grown indoor, (h) plantlets in soil grown outdoors (5 months old plants).

Expression of Pgli 79 in the Endosperm of Bg 250 Rice Variety

Genomic DNA was extracted from putative transgenic rice plants and analyzed by PCR using gliadin gene specific primers to confirm the presence of the transgene (Fig. 7). The expression of gliadin in rice seeds were confirmed by SDS-PAGE and western blot analysis (Fig. 8).

FIGURE 7.

Amplification of the 861 bp gliadin gene from T₀ plants: Lane 1 – 1 kb ladder (Invitrogen, USA), Lane 2 – genomic DNA of non-transformed rice plant leaves, Lanes 3,4,5,6,7,8,9 – genomic DNA of different putative transgenic rice leaves, Lane 10 - negative control (PCR water) and Lane 11 - Positive control (recombinant pCAMBIA vector containing gliadin gene).

FIGURE 8.

Identification of gliadin protein in transgenic rice flour using western blot analysis. Lane 1 - non-transgenic rice flour, Lane 2,3 - transgenic rice flour, Lane 4 - wheat flour and M - broad spectrum protein ladder stained with Coomassie blue R-250 (used in SDS-PAGE) aligned with the western blot. Red arrow indicates the expressed gliadin protein bands.

DISCUSSION

Gliadins represent a set of important seed-storage proteins in wheat, and both their composition and quantity significantly affect wheat flour quality.³³ Li and co-workers²⁷ reported variations in the number of α-helices and β-strands in different α-gliadins including variations in the amino acid residues of conserved α-helices and β-strands. However, the locations of α-helices and β-strands and core sequences were relatively conserved. α-gliadins were classified depending on the presence or absence of the relatively conserved β-strand (S) in the C-terminal unique domain II. Each type can be subdivided into eight groups based on the positions of the extra α-helix and β-strand. The clone pgli 79 had an extra α-helix HE₁ (Table 3). Our study also confirmed the fact that both pgli 70 & 79 have a conserved region at the N terminal repetitive domain. However, the secondary structure of pgli 70 & 79 were not similar. This could be due to the single amino acid variation observed at the N terminal domain of pgli 70 & 79. Previous studies by Xie et al. (2010)³⁴ and Li et al. (2014)²⁷ suggest that unique domains were the most important regions for the function of α-gliadins, whereas in some cases the glutamine repeats would also contribute. Generally, the presence of a long repetitive domain, a high proportion of glutamine residues, an extra cysteine residue in the primary structure and additional α-helices/β-strands in the secondary structure of the α-gliadin will improve the positive effect on gluten quality.^18,35–37

TABLE 3.

Secondary structure prediction of the two deduced α-gliadins.

α-gliadin	Secondary structure	A HE1	B H1	C H2 HE2 H3	D H4	E HE4 SE H5 S
Pgli 70	α helix	1	1	1 0 1	1	0 0 1 0
Pgli 79	α helix	0	1	1 0 1	1	0 0 1 0

The presence (1) and absence (0) of helices (H) and β strands (S) in different positions of the gliadin gene. A; N-terminal repetitive domain, B; polyglutamine domain I, C; unique domain I D; polyglutamine domain II and E; C-terminal unique domain II.

The six cysteine residues of pgli 70 were conserved in the two unique domains and form three intramolecular disulphide bonds, contributing to the structure of the gliadin protein. The pgli 79 gliadin with an odd number of cysteines (5) would, have one free cysteine after intramolecular disulphide bond formation. The free cysteine residue can participate in inter molecular disulfide bond formation.

An odd number of cysteine residues and the secondary structure consisting of six α-helices strongly suggest that pgli 79 is closely associated with the quality of common wheat cultivar Dacke to make bread. However, the present wheat cultivar Dacke has the full potential to induce the CD syndrome. In different parts of the world, approximately 0.5% to 1% people are affected with celiac disease. To address this concern, mutagenesis³⁸, RNAi technique³⁹ and genome editing⁴⁰ has being carried out to reduce/eliminate the celiac toxicity causing epitopes in wheat. Furthermore, it is also possible to custom synthesize the gene without the DNA sequence that codes for the toxic epitopes.

Interestingly, the α-gliadin gene was expressed in the transgenic rice seeds. It is the first report on the expression of the novel α-gliadin protein in transgenic rice. Agrobacterium mediated tissue culture method was used to generate transgenic plants in the present study.

Seven putative transgenic rice plants were generated on hygromycin selection, of which three plants were found to be positive for α-gliadin gene by the PCR (Fig. 7). The expression of the transgene was confirmed by SDS–PAGE and western blot analysis for two plants. A pale band of the expected size (~30 kDa) was observed in the western blot (Lane 2&3: Fig. 8). The possible reasons for pale bands could be either due to sub optimal extraction of the desired protein or due to low expression of the protein in the transgenic seeds. It has been reported that the copy number of transgenes, position effects or DNA rearrangements may lead to differential expression of transgenic proteins.⁴¹

Non-specific bands were observed in transgenic rice seeds (Lane 2&3: Fig. 8) in the western blot. These non-specific bands were also observed in non-transgenic rice seeds (Lane 1: Fig. 8). This is most probably due to cross reactivity of other rice proteins that may have co-purified with gliadin in the extraction procedure.^42,43

The findings of this study could potentially be used to produce wheat-like rice. To achieve this goal, all three genes (gliadin, LMW and HMW glutenin) have to be expressed in a single rice plant. This would require the generation of T₁, T₂, T₃, T_{4 …} plants to identify homozygous lines. Once the homozygous lines are identified, they can be artificially crossed for generating transgenic rice line expressing all three genes.

CONCLUSION

Five α-gliadin genes were amplified from the wheat cultivar Dacke and it was compared with α-gliadins from other wheat species. The α-gliadins from the Dacke wheat variety contained low numbers of toxic epitopes, an odd number of cysteine residues and six α-helices in the secondary structure that might be beneficial to wheat flour quality. Therefore, the expression of α-gliadin in rice endosperm is a promising application to improve the gluten in rice endosperm.

Funding Statement

This work was supported by the National Science Foundation [RG/2010/BT/04].