Γ-gliadin Gene Research Articles

CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole-genome sequencing of an edited line showed mutation or deletion of nearly all ω-gliadin and half of the γ-gliadin gene copies and confirmed the lack of editing in the α/β-gliadin genes. The estimated 75% and 64% reduction in ω- and γ-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to a non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the ω- and γ-gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

A new target for improving wheat end-use quality.

This article is a Commentary on Liu et al. (2023), 239: 87–101.

An elite γ-gliadin allele improves end-use quality in wheat.

Gluten is composed of glutenins and gliadins and determines the viscoelastic properties of dough and end-use quality in wheat (Triticum aestivum L.). Gliadins are important for wheat end-use traits, but the contribution of individual gliadin genes is unclear, since gliadins are encoded by a complex, multigenic family, including many pseudogenes. We used CRISPR/Cas9-mediated gene editing and map-based cloning to investigate the contribution of the γ-gliadin genes annotated in the wheat cultivar 'Fielder', showing that Gli-γ1-1D and Gli-γ2-1B account for most of the γ-gliadin accumulation. The impaired activity of only two γ-gliadin genes in knockout mutants improved end-use quality and reduced gluten epitopes associated with celiac disease (CD). Furthermore, we identified an elite haplotype of Gli-γ1-1D linked to higher end-use quality in a wheat germplasm collection and developed a molecular marker for this allele for marker-assisted selection. Our findings provide information and tools for biotechnology-based and classical breeding programs aimed at improving wheat end-use quality.

Unprocessed wheat γ-gliadin reduces gluten accumulation associated with the endoplasmic reticulum stress and elevated cell death.

Summary Along with increasing demands for high yield, elite processing quality and improved nutrient value in wheat, concerns have emerged around the effects of gluten in wheat‐based foods on human health. However, knowledge of the mechanisms regulating gluten accumulation remains largely unexplored.Here we report the identification and characterization of a wheat low gluten protein 1 (lgp1) mutant that shows extremely low levels of gliadins and glutenins.The lgp1 mutation in a single γ‐gliadin gene causes defective signal peptide cleavage, resulting in the accumulation of an excessive amount of unprocessed γ‐gliadin and a reduced level of gluten, which alters the endoplasmic reticulum (ER) structure, forms the autophagosome‐like structures, leads to the delivery of seed storage proteins to the extracellular space and causes a reduction in starch biosynthesis. Physiologically, these effects trigger ER stress and cell death.This study unravels a unique mechanism that unprocessed γ‐gliadin reduces gluten accumulation associated with ER stress and elevated cell death in wheat. Moreover, the reduced gluten level in the lgp1 mutant makes it a good candidate for specific diets for patients with diabetes or kidney diease.

A Combinatorial Q-Locus and Tubulin-Based Polymorphism (TBP) Approach Helps in Discriminating Triticum Species.

The simple and straightforward recognition of Triticum species is not an easy task due to their complex genetic origins. To provide a recommendation, we have compared the performance of different PCR-based methods relying on the discrimination ability of the Q- and γ-gliadin (GAG56D) genes, as well as TBP (Tubulin-Based Polymorphism), a method based on the multiple amplification of genes of the β-tubulin family. Among these approaches, the PCR-RFLP (Restriction Fragment Length Polymorphism) assay based on a single-nucleotide polymorphism (SNP) present in the Q gene is the only one capable of fully discerning hexaploid spelt and common wheat species, while both γ-gliadin and TBP fail with similar error frequencies. The Q-locus assay results in the attainment of either a single fragment or a doublet, depending on the presence of a suitable restriction site, which is affected by the mutation. This dual pattern of resolution limits both the diagnostic effectiveness, when additional Triticum species are assayed and compared to each other, and its usefulness, when commercially available flours are analyzed. These limitations are overtaken by flanking the Q-locus assay with the TBP analysis. In this way, almost all of the Triticum species can be accurately identified.

Duplex droplet digital PCR (ddPCR) method for the quantification of common wheat (Triticum aestivum) in spelt (Triticum spelta)

A duplex droplet digital PCR (ddPCR) is described for the detection and quantification of contaminations by common wheat (Triticum aestivum) in food products made from spelt (Triticum spelta). A single nucleotide polymorphism (SNP) in the locus Q, as well as a short sequence of the γ-gliadin gene of the wheat genome are exploited for the development of our duplex ddPCR method. Minimal variations of DNA sequences causing different probe hydrolysis efficiencies during PCR amplification can be detected and evaluated in ddPCR. By utilizing the SNP in the locus Q, all 84 tested spelt cultivars could unambiguously discriminated from common wheat cultivars. Discrimination using the γ-gliadin gene sequence could be achieved for 10 of 84 spelt cultivars. Additionally, we could show that impurities caused by common wheat can also be detected in einkorn wheat (T. monococcum) and in emmer (T. dicoccon).

Genome-Wide Identification, Characterization and Expression Pattern Analysis of the γ-Gliadin Gene Family in the Durum Wheat (Triticum durum Desf.) Cultivar Svevo.

Very recently, the genome of the modern durum wheat cv. Svevo was fully sequenced, and its assembly is publicly available. So, we exploited the opportunity to carry out an in-depth study for the systematic characterization of the γ-gliadin gene family in the cv. Svevo by combining a bioinformatic approach with transcript and protein analysis. We found that the γ-gliadin family consists of nine genes that include seven functional genes and two pseudogenes. Three genes, Gli-γ1a, Gli-γ3a and Gli-γ4a, and the pseudogene Gli-γ2a* mapped on the A genome, whereas the remaining four genes, Gli-γ1b, Gli-γ2b, Gli-γ3b and Gli-γ5b, and the pseudogene Gli-γ4b* mapped on the B genome. The functional γ-gliadins presented all six domains and eight-cysteine residues typical of γ-gliadins. The Gli-γ1b also presented an additional cysteine that could possibly have a role in the formation of the gluten network through binding to HMW glutenins. The γ-gliadins from the A and B genome differed in their celiac disease (CD) epitope content and composition, with the γ-gliadins from the B genome showing the highest frequency of CD epitopes. In all the cases, almost all the CD epitopes clustered in the central region of the γ-gliadin proteins. Transcript analysis during seed development revealed that all the functional γ-gliadin genes were expressed with a similar pattern, although significant differences in the transcript levels were observed among individual genes that were sometimes more than 60-fold. A progressive accumulation of the γ-gliadin fraction was observed in the ripening seeds that reached 34% of the total gliadin fraction at harvest maturity. We believe that the insights generated in the present study could aid further studies on gliadin protein functions and future breeding programs aimed at the selection of new healthier durum wheat genotypes.

Promoter DNA hypermethylation of TaGli-γ-2.1 positively regulates gluten strength in bread wheat

IntroductionGliadins are the major components of gluten proteins with vital roles on properties of end-use wheat product and health-relate quality of wheat. However, the function and regulation mechanisms of γ-gliadin genes remain unclear. ObjectivesDissect the effect of DNA methylation in the promoter of γ-gliadin gene on its expression level and gluten strength of wheat. MethodsThe prokaryotic expression and reduction–oxidation reactions were performed to identify the effect of TaGli-γ-2.1 on dough strength. Bisulfite analysis and 5-Aza-2′-deoxycytidine treatment were used to verify the regulation of TaGli-γ-2.1 expression by pTaGli-γ-2.1 methylation. The content of gluten proteins composition was measured by RP-HPLC, and the gluten strength was measured by Gluten Index and Farinograph. ResultsTaGli-γ-2.1 was classified into a subgroup of γ-gliadin multigene family and was preferentially expressed in the later period of grain filling. Addition of TaGli-γ-2.1 protein fragment into strong gluten wheat flour significantly decreased the stability time. Hypermethylation of three CG loci of pTaGli-γ-2.1 conferred to lower TaGli-γ-2.1 expression. Treatment with 5-Aza-2′-deoxycytidine in seeds of strong gluten wheat varieties increased the expression levels of TaGli-γ-2.1. Furthermore, the accumulations of gliadin and γ-gliadin were significantly decreased in hypermethylated wheat varieties, corresponding with the increasing of gluten index and dough stability time. ConclusionEpigenetic modification of pTaGli-γ-2.1 affected gluten strength by modulating the proportion of gluten proteins. Hypermethylation of pTaGli-γ-2.1 is a novel genetic resource for enhancing gluten strength in wheat quality breeding.

Comparison of alleles at the Gli-1 loci of common wheat by means of two-dimensional electrophoresis of gliadin and RFLP analysis

Allelic variants of the Gli-1 locus is known to control groups (blocks) of gliadin polypeptides (gliadins). Some allelic variants of blocks that differ in the electrophoretic (acid gel) mobility (EM) of only one gliadin of the block were compared using two-dimensional electrophoresis (SDS-PAGE) and the RFLP procedure. It was found that, in these pairs of similar alleles (Gli-B1f, Gli-B1s, and Gli-D1a as compared with Gli-B1e, Gli-B1n, and Gli-D1c, respectively), faster γ-gliadin had smaller molecular weight (MW). Alleles at the Gli-A1 locus (Gli-A1j, Gli-A1i, Gli-A1a, Gli-A1k, and Gli-A1f) differ in the EM of the γ-gliadin so that Gli-A1j controls the slowest γ-gliadin and Gli-A1f controls the fastest one. We found that, in this order of alleles, faster γ-gliadin always had smaller MW. It was suggested that similar alleles might arise from one another by spontaneous mutations changing the number of repeating sequences or length of the polyglutamine domain present in the γ-gliadin gene thereby influencing MW and EM of encoding polypeptide. Other mechanisms of the mutational appearance of new alleles were found earlier by comparison of allele pairs: Gli-D1a and Gli-D1k (gene silencing) and Gli-D1b and Gli-D1d (gene amplification). We discovered contrasting families of alleles at the Gli-B1 and at the Gli-D1 loci and also two variants of apparently the same allele Gli-D1a that differed in the number of encoded ω-gliadins. Families of alleles at one locus of T. aestivum might inherit from different genotypes of corresponding diploid donor, as we suggested earlier.

Cloning and characterization of novel γ-gliadin genes from Aegilops markgrafii in relation to evolution and wheat breeding

Gliadins are the major components of storage proteins in wheat and play an important role in determining the extensibility properties of dough. In the present work, six novel full-length γ-gliadin genes were cloned from the C genome of Aegilops markgrafii using a PCR-based strategy. Analysis of the deduced amino acid sequences showed that the cloned genes had primary structures that were similar, but not identical, to published γ-gliadins from other wheat-related species. The lengths of the open reading frames (ORFs) ranged from 909 to 963bp, and the repetitive and glutamine-rich domains were mainly responsible for the size of the proteins. An extra cysteine residue was present in the repetitive domain of sequence JX566513. All amino acid sequences of γ-gliadin genes from Ae. markgrafii were searched for the five peptides identified as T cell stimulatory epitopes in celiac disease (CD) patients. Peptide Gliγ-3 was present in sequences JX566513 and JX566514. Peptide Gliγ-5 was present only in JX566513. The other γ-gliadins contained no toxic epitopes. These results provide information to better understand the use of Ae. markgrafii in wheat breeding and the evolutionary relationship of the γ-gliadin genes in Ae. markgrafii and other Triticeae species.

Genome-, Transcriptome- and Proteome-Wide Analyses of the Gliadin Gene Families in Triticum urartu.

Gliadins are the major components of storage proteins in wheat grains, and they play an essential role in the dough extensibility and nutritional quality of flour. Because of the large number of the gliadin family members, the high level of sequence identity, and the lack of abundant genomic data for Triticum species, identifying the full complement of gliadin family genes in hexaploid wheat remains challenging. Triticum urartu is a wild diploid wheat species and considered the A-genome donor of polyploid wheat species. The accession PI428198 (G1812) was chosen to determine the complete composition of the gliadin gene families in the wheat A-genome using the available draft genome. Using a PCR-based cloning strategy for genomic DNA and mRNA as well as a bioinformatics analysis of genomic sequence data, 28 gliadin genes were characterized. Of these genes, 23 were α-gliadin genes, three were γ-gliadin genes and two were ω-gliadin genes. An RNA sequencing (RNA-Seq) survey of the dynamic expression patterns of gliadin genes revealed that their synthesis in immature grains began prior to 10 days post-anthesis (DPA), peaked at 15 DPA and gradually decreased at 20 DPA. The accumulation of proteins encoded by 16 of the expressed gliadin genes was further verified and quantified using proteomic methods. The phylogenetic analysis demonstrated that the homologs of these α-gliadin genes were present in tetraploid and hexaploid wheat, which was consistent with T. urartu being the A-genome progenitor species. This study presents a systematic investigation of the gliadin gene families in T. urartu that spans the genome, transcriptome and proteome, and it provides new information to better understand the molecular structure, expression profiles and evolution of the gliadin genes in T. urartu and common wheat.

Identification of a Group of Novel γ-Gliadin Genes

γ-Gliadins are an important component of wheat seed storage proteins. Four novel γ-gliadin genes (Gli-ng1 to Gli-ng4) were cloned from wheat (Triticum aestivum) and Aegilops species. The novel γ-gliadins were much smaller in molecular size when compared to the typical γ-gliadins, which was caused by deletion of the non-repetitive domain, glutamine-rich region, 3' part of the repetitive domain, and 5' part of the C-terminal, possibly due to illegitimate recombination between the repetitive domain and the C-terminal. As a result, Gli-ng1 and Gli-ng4 only contained two and three cysteine residues, respectively. Gli-ng1, as the representative of novel γ-gliadin genes, has been sub-cloned into an Escherichia coli expression system. SDS-PAGE indicated that the both cysteine residues of Gli-ng1 could participate in the formation of intermolecular disulphide bonds in vitro. Successful cloning of Gli-ng1 from seed cDNA of T. aestivum cv. Chinese Spring suggested that these novel γ-gliadin genes were normally transcribed during the development of seeds. Phylogenic analysis indicated that the four novel γ-gliadin genes had a closer relationship with those from the B (S) genome of wheat.

The gene space in wheat: the complete γ-gliadin gene family from the wheat cultivar Chinese Spring

The complete set of unique γ-gliadin genes is described for the wheat cultivar Chinese Spring using a combination of expressed sequence tag (EST) and Roche 454 DNA sequences. Assemblies of Chinese Spring ESTs yielded 11 different γ-gliadin gene sequences. Two of the sequences encode identical polypeptides and are assumed to be the result of a recent gene duplication. One gene has a 3' coding mutation that changes the reading frame in the final eight codons. A second assembly of Chinese Spring γ-gliadin sequences was generated using Roche 454 total genomic DNA sequences. The 454 assembly confirmed the same 11 active genes as the EST assembly plus two pseudogenes not represented by ESTs. These 13 γ-gliadin sequences represent the complete unique set of γ-gliadin genes for cv Chinese Spring, although not ruled out are additional genes that are exact duplications of these 13 genes. A comparison with the ESTs of two other hexaploid cultivars (Butte 86 and Recital) finds that the most active genes are present in all three cultivars, with exceptions likely due to too few ESTs for detection in Butte 86 and Recital. A comparison of the numbers of ESTs per gene indicates differential levels of expression within the γ-gliadin gene family. Genome assignments were made for 6 of the 13 Chinese Spring γ-gliadin genes, i.e., one assignment from a match to two γ-gliadin genes found within a tetraploid wheat A genome BAC and four genes that match four distinct γ-gliadin sequences assembled from Roche 454 sequences from Aegilops tauschii, the hexaploid wheat D-genome ancestor.

Isolation and phylogenetic analysis of novel γ-gliadin genes in genus Dasypyrum

As the most ancient member of the wheat gluten family, the γ-gliadin genes are suitable for phylogenetic analysis among wheat and related species. Species in the grass genus Dasypyrum have been widely used for wheat cross breeding. However, the genomic relationships among Dasypyrum species have been little studied. We isolated 22 novel γ-gliadin gene sequences, among which 10 are putatively functional. The open reading frame lengths of these sequences range from 642 to 933 bp, and these putative proteins consist of five domains. Phylogenetic analyses showed that all Dasypyrum γ-gliadin gene sequences clustered in a large group; D. villosum and tetraploid D. breviaristatum γ-gliadin gene sequences clustered in a subgroup, while diploid D. breviaristatum γ-gliadin gene sequences clustered at the edge of the subgroup. All of the Dasypyrum γ-gliadin gene sequences were absent in three major T cell-stimulatory epitopes binding to HLA-DQ2/8 in celiac disease patients. Based on the phylogenetic analyses, we suggest that D. villosum and tetraploid D. breviaristatum evolved in parallel from a diploid ancestor D. breviaristatum.

A novel family of γ-gliadin genes are highly regulated by nitrogen supply in developing wheat grain

Six wheat cultivars were grown at Rothamsted (UK) with three levels of nitrogen fertilizer (100, 200 and 350kg N/ha) in 2009 and 2010. Gene expression in developing caryopses at 21 days post-anthesis (DPA) was profiled using the Affymetrix Wheat GeneChip®. Four of 105 transcripts which were significantly upregulated by nitrogen level were annotated as γ-3 hordein and the identification of corresponding expressed sequence tags showed that they differed in sequence from previously described (typical) γ-gliadins and represented a novel form of γ-gliadin. Real-time reverse transcriptase PCR at 14, 21, 28 and 35 DPA revealed that this transcript was most abundant and most responsive to nitrogen at 21 DPA. Four novel γ-gliadin genes were isolated by PCR amplification from wheat cv. Hereward and the related species Aegilops tauschii and Triticum monococcum while three were assembled from the genomic sequence database of wheat cv. Chinese Spring (www.cerealsdb.uk.net). Comparison of the deduced amino acid sequences of the seven genes showed that they shared only 44.4–46.0% identity with the sequence of a typical γ-gliadin (accession number EF15018), but 61.8–68.3% identity with the sequence of γ-3 hordein from the wild barley species Hordeum chilense (AY338065). The novel γ-gliadin genes were localized to the group 1 chromosomes (1A, 1B, 1D).

Molecular characterization and dynamic expression patterns of two types of γ-gliadin genes from Aegilops and Triticum species

Gliadins were the major components of wheat storage proteins and determine the extensibility properties of gluten dough. In this work, 19 new full-length γ-gliadin genes were isolated from various Aegilops and Triticum species. Sequence characterization showed that a specific octapeptide and celiac disease (CD)-toxic epitope Gliγ-3 (VQGQGIIQPQQPAQL) were present in the rich glutamine domain and C-terminal non-repetitive domain, respectively. Based on the sequence features of both peptides, a new classification system for γ-gliadin gene family was established, in which γ-gliadins were classified into two types (types I and II) with each consisting of two groups. An uneven distribution of different types and groups of γ-gliadin genes was exhibited among 11 Aegilops and Triticum genomes. Phylogenetic analysis revealed that types I and II genes diverged at about 14 MYA while the divergence of 4 γ-gliadin group genes occurred at around 10 MYA almost simultaneously. The γ-gliadin genes from S(l) and B genomes displayed a different transcriptional expression pattern during grain development, and rapid increasing of gliadin mRNA and proteins occurred at 15-20 DPA. In addition, genome-specific variations of CD-toxic epitopes among Aegilops and Triticum genomes were found. The A genome and its related progenitor genomes A(u) and A(m) had fewer CD epitopes than other genomes, suggesting that these genomes might be valuable gene resources to remove CD toxic peptides for wheat quality improvement.

Use of Polymorphisms in the γ-Gliadin Gene of Spelt and Wheat as a Tool for Authenticity Control

Partial sequencing of the γ-gliadin gene of 62 spelt and 14 soft wheat cultivars was performed. Fifty-six of the 62 spelt cultivars and 13 of the 14 soft wheat cultivars were shown to exhibit the typical spelt or soft wheat γ-gliadin sequence, respectively. Exceptions were ascribed to crossbreeding of soft wheat and spelt. Using the typical soft wheat γ-gliadin sequence, two alternative DNA-based analytical methods were developed for the detection and quantification of spelt flour "adulteration" with soft wheat. A simple and fast detection of soft wheat in spelt flours could be achieved by restriction fragment length (RFLP) analysis. In combination with lab-on-a-chip capillary gel electrophoresis (LOC-CE) the soft wheat proportion could be estimated. Heteroduplex formation served as additional confirmation for the presence of spelt besides soft wheat. Hence, RFLP-LOC-CE constitutes a perfect analysis tool for the quality control of cereal seeds and pure cultivars. A precise quantification of soft wheat "adulterations" in spelt flour down to 1% could be achieved by the developed real-time PCR method. The calibration parameters of the real-time PCR assay fulfilled the minimum performance requirements of the European Network of GMO (genetically modified organisms) Laboratories (ENGL).

Cloning, Prokaryotic Expression, and Functional Testing of a γ-Gliadin Gene from Wheat Cultivar Shaan 253

The γ-gliadin gene (GenBank accession No. GQ857626) was isolated from a wheat (Triticum aestivum) cultivar with a hight quality Shaan 253, using inductuion a set of genome specific primers. The deduced amino acid sequence of GQ857626 exhibited an additional cysteine residue in the region II of mature γ-gliadin subunits compared to the previous report. A prokaryotic expression vector was constructed to express GQ857626 in the host bacterium Escherichia coli Rosetta-gami B(DE3). The SDS-PAGE and Western blot analysis confirmed that the fusion protein was expressed by induction of 0.1 mmol L^(-1) isopropyl-β-D-thiogalactoside (IPTG), and its actual molecular weight was about 50 kD nearly equal to the predicted. After separation and purification using HisTrap HP affinity chromatography, the function of fusion protein was tested by means of micro 4 g farinograph. When the subunit of GQ857626 was integrated into the control flour through oxidation-reduction reaction, many important farinograph parameters changed drastically, such as development time, dough stabilizing time, degree of softening. Although the gluten strength of the control flour could be improved in some degree, the subunit of GQ857626 showed a negative effect on the rheological properties of wheat flour.

Characterization of two novel γ-gliadin genes encoded by K genome of Crithopsis delileana and evolution analysis with those from Triticeae

By acid polyacrylamide gel electrophoresis (A-PAGE) analysis, it was indicated that the electrophoresis mobility of gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) had obvious difference with those from common wheat in α, γ and ω region. Using homologous primers, two γ-gliadin genes (gli-Kr1 and gli-Kr2) were isolated from C. delileana, which had been deposited in the GenBank under accession numbers EU283818 and EU283821, respectively. Two γ -gliadin genes of C. delileana had the similar primary structures to the corresponding gene sequences from other wheat related species. The differences were mainly resulted from substitutions, insertions and deletions involving single amino acid residues or motifs of γ-gliadins. The repetitive domains of gli-Kr1 and gli-Kr2 from C. delileana are shorter than most of other sequences. By the alignment of γ-gliadin genes from A, B, D, Am, Au, S, Sl, Ssh, Ss and Sb genomes of Triticum and Aegilops, R genome of Secale (γ-secalin), Ee genome of Lophopyrum and K genome of Crithopsis in Triticeae, phylogenetic analysis indicated that two γ-gliadin genes of C. delileana could be clustered together with a γ-gliadin genefrom Ssh genome of Aegilops by an interior paralleled branch. It was the first time that the γ-gliadin genes encoded by K genome of C. delileana were characterized. These could offer precious information for better understanding the qualities associated with gliadins, the response in coeliac disease and studying the evolutionary relationship of gliadins in Triticeae.

Gene Cloning and Sequence Analysis of γ-Gliadin Genes from Wheat Cultivar Shaan 253

Eight γ-gliadin genes (GenBank accession number GQ87170 to GQ87177) were cloned from wheat (Triticum aestivum L.) cultivar Shaan 253 using three sets of specific primers, which were designed according to the known γ-gliadin gene family. All of the eight genes possess the typical structure of γ-gliadin subunits and many sequences of insent/indel in the repeat region. The deduced amino acid sequences showed that GQ871771 is a pseudogene, and nine cysteine residues existed in GQ871770, GQ871772, GQ871776, and GQ87177. Promoter sequences analysis indicated that there were six SNPs in endosperm box of GQ871770, GQ871772, GQ87174, and GQ871776, and two of them appeared in the GCN4-like motif. A model for endosperm box of storage protein with better representation was constructed using the online tool WebLogo3. Phylogenetic analysis also confirmed that the cloned sequences belong to γ-gliadin gene family.