Generation of transgenic rice with reduced content of major and novel high molecular weight allergens
© Ogo et al.; licensee springer. 2014
Received: 1 May 2014
Accepted: 31 July 2014
Published: 29 August 2014
Rice seed proteins contain antigens that provoke allergic responses in some individuals with food allergy, particularly in those with cereal allergy, and these antigens can elicit clinical symptoms such as eczema and dermatitis. We previously generated transgenic rice with reduced accumulation of the three major allergens, which dramatically reduced the level of IgE binding from patients’ sera. However, the transgenic rice still possesses allergenic reactivity. Recently, two globulin-like proteins were identified as candidates of novel high molecular weight (HMW) IgE-binding proteins that cause rice allergy.
We identified a glucosidase family encoded by four genes as novel HMW rice allergens based on IgE antibody reactivity from individuals with allergy to rice. To further reduce allergenicity, we generated transgenic rice with reduced accumulation of these HMW allergens. We crossed the rice with reduced HMW allergens and with reduced major allergens, and all major and HMW allergens were substantially reduced in the progeny of the crossed rice. Allergen suppression did not significantly alter accumulation patterns of seed storage proteins and protein folding enzymes. The sera of a portion of patients showed low IgE-binding to the crossed line, suggesting that the crossed line is effective for a portion of patients who are allergic to proteins other than major allergens.
The transgenic rice with reduced levels of all major and HMW allergens is thought to be an option for a portion of allergy patients with hypersensitive responses to various kinds of rice allergens.
Rice is a major cereal food consumed by more than half of the world population. The prevalence of IgE-mediated rice allergy is approximately 10% in atopic subjects. Symptoms of rice allergy include atopic dermatitis, eczema, and food-protein-induced enterocolitis syndrome (Hoffman ; Shibasaki et al., ; Ikezawa et al., ; Sicherer et al. ; Uchio et al., ; Mehr et al., ). Multiple rice seed proteins are responsible for rice allergy (Urisu et al., ). Among them, α-globulin (26 kDa), β-glyoxalase I (33 kDa), and α-amylase/trypsin inhibitor (14–16 kDa) were identified as major rice allergens based on recognition by IgE from individuals with food allergy (Alvarez et al., ; Limas et al., ; Usui et al., ; Matsuda et al., ). The 14–16 kDa α-amylase/trypsin inhibitors constitute a multigene family, whereas the 26 and 33 kDa allergens are encoded by single-copy genes. These allergens strongly react with IgE antibody in sera from many individuals with rice allergy, and caused eczematous and atopic dermatitis (Urisu et al., ).
Avoidance of food containing allergens is one of the most important therapeutic strategies for those with food allergy. Allergens have been removed from rice by several processing technologies such as enzymatic digestion, alkaline hydrolysis, and high hydrostatic pressure (Watanabe et al., [1990a], [1990b]; Kato et al., ). Some of these processes have been commercialized to produce low-allergen rice in Japan; however, the taste quality of processed rice is reduced by these chemical, enzymatic, or physical treatments, and the treatments are costly. Therefore, it is important to develop a cost-effective means to produce low-allergen (hypo-allergenic) rice with good taste. We previously generated hypo-allergenic transgenic rice, in which the levels of major seed allergen genes (26 kDa, 33 kDa, and 14–16 kDa allergens) were suppressed (Wakasa et al., [2011a]). To suppress the major allergen levels in rice grains, we first found a mutant in the Koshihikari background that lacked the 26 kDa allergen (GbN-1). Then, 33 kDa and 14–16 kDa allergen levels were suppressed by RNA interference (RNAi) using GbN-1 as a host rice. In the transgenic line, the content of the three major allergens was remarkably reduced to a very faint level. IgE binding of patients’ sera to the transgenic rice seed with reduced levels of major allergens was substantially lower compared with that of non-transgenic (NT) Koshihikari (Wakasa et al., [2011a]). However, some individuals retained allergenic reactivity to the transgenic rice. These results indicate that additional allergens are present in rice, which must be removed for the optimum health of individuals with a wide variety of allergies to rice.
Several major rice allergens were identified in previous studies. Urisu et al. () showed that IgE from patients with rice allergy detected several allergenic proteins with high molecular weight (HMW), which have not yet been identified. Two globulin-like proteins of 52 kDa and 63 kDa, which correspond to the HMW allergenic proteins, have recently been identified as novel IgE-binding proteins. These globulin-like proteins are candidates for rice allergens based on recognition by IgE from patients with rice allergy (Satoh et al., ). The 52 kDa and 63 kDa proteins are strongly expressed in rice seed, and are thought to be major causes of rice allergy. Here, we identified a multigene glucosidase family as novel IgE-binding HMW proteins, which are also candidates for rice allergens. We suppressed the levels of these HMW allergens by RNAi and crossed the RNAi rice with the transgenic rice with reduced levels of the major allergens. The allergen-reduced transgenic rice is a promising candidate for generating hypo-allergenic rice.
Results and discussion
Identification of HMW rice allergens
Identification of IgE-binding rice proteins by MALDI-TOF MS/MS
Sequence coverage (%)
Sequence coverage (%)
Generation of transgenic rice with reduced levels of the HMW allergens
Western blot of 52 kDa and 63 kDa globulins
Real-time PCR of ONG1, ONG2&3, and ONG4
We attempted to generate antibodies against ONG1, ONG2&3, and ONG4; however, strongly reactive antibodies to ONGs were not produced. Therefore, mRNA expression of ONG1, ONG2&3, and ONG4 in transgenic rice seeds was investigated by real-time PCR. We extracted total RNA from transgenic rice seeds with endosperm- and embryo-specific suppression of the HMW allergens and performed real-time PCR. The mRNA levels of ONG1, ONG2&3, and ONG4 were substantially reduced in a few lines (Figure 3C). According to the Western blot of 52 kDa and 63 kDa globulins and real-time PCR of ONGs, we identified a line in which the levels of 52 kDa globulin, 63 kDa globulin, ONG1, ONG2&3, and ONG4 were substantially suppressed in endosperm and embryo.
Crossing transgenic rice lines with reduced levels of major and HMW allergens
Target gene suppression efficiency in transgenic rice lines
Total F2 seed
26 & 52 kDa
Among both 26 kDa and 52 kDa suppressed seeds,
Allergenic potential of the rice lines with reduced levels of the major and HMW allergens
Seed characteristics in rice lines with reduced levels of the major and HMW allergens
Seed characteristics in the transgenic rice lines
Protein content (%)
4.71 ± 0.24
2.65 ± 0.09
1.84 ± 0.04
17.9 ± 0.5
13.5 ± 0.6
4.65 ± 0.18
2.27 ± 0.30
1.75 ± 0.09
14.0 ± 0.7
14.9 ± 0.9
4.68 ± 0.08
2.68 ± 0.11
1.78 ± 0.05
16.9 ± 0.6
13.6 ± 1.2
Major x HMW
4.75 ± 0.21
2.44 ± 0.20
1.78 ± 0.04
15.0 ± 0.6
14.5 ± 1.3
Once the safety evaluation of the transgenic rice is performed, Major x HMW lines can be utilized by individuals with a rice allergy whose serum IgE recognizes these allergens. The transgenic rice approach has several advantages over conventional hypo-allergenic rice produced by processing with enzymatic and physical treatments. Our hypo-allergenic rice was generated using the highly edible Koshihikari variety, and the transgenic product is cost-effective. The seed quality of Major x HMW lines can be improved by backcrossing with Koshihikari. Major x HMW lines are thought to be acceptable as a hypo-allergenic rice for a portion of patients with rice allergies.
We identified a glucosidase family as novel rice HMW allergens. We successfully generated transgenic rice with reduced content of the major and the HMW allergens, which showed reduced IgE binding to sera of a portion of rice allergy patients who are allergic to proteins other than the major allergens. These transgenic rice are thought to be an option for a portion of allergy patients with hypersensitive responses to various kinds of rice allergens.
SDS-PAGE and Western blot for identification of HMW allergens
Seed proteins (the albumin-globulin fraction) were extracted from rice seeds (Oryza sativa cv. Koshihikari), as described previously (Wakasa et al., [2011a]). The extracted proteins were separated by SDS-PAGE (10% acrylamide), followed by Western blot using the serum IgE from individual patients, as described previously (Usui et al., ; Hirano et al., ). Protein bands bound with IgE were immunologically detected with peroxidase-labeled anti-human IgE antibody (Nordic immunological Laboratories, Susteren, Netherlands) and a chemiluminescence detection kit (GE Healthcare Biosciences, Piscataway, NJ, USA).
Patient serum samples
Blood was collected from patients with suspected allergic disorders including food allergy under medical treatment at the Hospital of Fujita Health University. Informed consent was obtained from the patients or guardians of infants or child subjects. Serum specimens were prepared from fresh blood and kept as 50% glycerol mixture at −30°C until use. This study using patient serum specimens was approved by the Ethics Committee of Fujita Health University School of Medicine. Twenty-four serum specimens with relatively high IgE-binding to the rice seed albumin/globulin fraction were selected as rice-positive specimens, whereas three specimens with relatively low IgE-binding were selected as control specimens.
Protein identification by MS/MS analysis
Protein bands detected by Coomassie brilliant blue staining were excised, in-gel digested with trypsin, and extracted according to the manufacturer’s instructions. MS and MS/MS (tandem MS) analyses were performed using a MALDI–TOF/TOF mass spectrometer (4700 Proteomics Analyzer; Applied Biosystems, CA, USA), as described previously (Okumura et al., ). The obtained MS and MS/MS data were analyzed using Mascot Daemon data analysis software (Matrix Science; http://www.matrixscience.com/).
Immuno-dot-blot analysis of rice seed proteins for human IgE
IgE binding of patients’ IgE was analyzed by dot-blotting for limited amounts of serum specimens as described previously (Wakasa et al., [2011a]). Briefly, an aliquot of each rice seed extract, which was equivalent to 100 μg of milled rice seeds, was spotted on a small piece of nitrocellulose membrane, blocked with gelatin solution, and then incubated with 100-fold diluted human serum specimen. The IgE bound to the rice proteins on the membrane was detected using HRP-labeled secondary antibody and a chemiluminescence HRP substrate, and the chemiluminescence intensity was quantified using NIH Image-J.
Plant material and growth conditions
Rice plants (Oryza sativa cv. Koshihikari) were grown at 25°C/20°C (12 h/12 h day/night cycles) in 12 cm diameter pots containing a commercial soil mixture (Bonsol No. 1; Sumitomo Chemicals, Osaka, Japan) with 14-14-14 chemical fertilizer. The host of the line with reduced levels of major allergens is a seed storage protein mutant named GbN-1 (cv. Koshihikari background), which lack the 26 kDa allergen (Iida et al., ).
Binary vectors were constructed for Agrobacterium-mediated transformation using the MultiSite Gateway LR clonase reaction (Invitrogen), as described previously (Wakasa et al., ). Briefly, gene cassettes consisting of an endosperm-specific promoter [16 kDa prolamin promoter (0.93 kb), 13 kDa prolamin promoter (1.23 kb), and GluB-1 promoter (2.4 kb)], the inverted repeat structure of sense and antisense fragments from the coding region of ONG1-4, 52 kDa globulin (0.76 kb), or 63 kDa globulin (0.92 kb) separated by the rice oryzasin1 intron (0.99 kb) (Asakura et al., ; Kuroda et al., ), and a terminator [16 kDa prolamin (0.62 kb) terminator, 13 kDa prolamin terminator (0.18 kb), or GluB-1 terminator (0.6 kb)] were inserted into the Gateway entry clones pKS221 MCS, pKS 4–1 MCS and pKS 2–3 MCS. The homologous region of ONG1 and ONG2&3 (0.72 kb), and part of ONG4 (0.7 kb) were fused and used as an inverted repeat structure of sense and antisense fragments of ONGs. These gene cassettes were introduced into pCSP mALS 43GW using MultiSite Gateway LR Clonase II Plus Enzyme Mix (Invitrogen) (Figure 2). The binary vector plasmids were introduced into the Koshihikari cv. via Agrobacterium-mediated transformation, as described previously (Wakasa et al., ).
Protein extraction and Western blot analysis of the reduced allergen rice
Total protein extraction and Western blot were performed as described previously (Wakasa et al., [2011a]). Antibodies to 26 kDa, 33 kDa, and 14–16 kDa allergens, glutelins (GluA, GluB, and GluC), RM1, RM2, RM4, RM9, 16 kDa prolamin, 10 kDa prolamin, OsBiP1, OsPDIL1;1, and calnexin were previously prepared in our laboratory (Takagi et al., , Yasuda et al., ; Wakasa et al., [2011a], [2011b]). The MH2-RRGEREEEDERRRHG –OH and MH2-SGEDRRRETSLRRC -OH peptides derived from 52 kDa (Os03g0793700) and 63 kDa (Os03g0663800) allergens, respectively, were synthesized and used to raise anti-52 and 63 kDa allergens polyclonal antibody in a rabbit (Scrum Inc., Tokyo, Japan). Seed protein content was measured by an RC DC protein assay kit (Bio-Rad), as described in the manufacturer’s protocol.
RNA preparation and real-time PCR
Total RNA was prepared from mature seeds of NT and transgenic rice plants, as described previously (Yasuda et al., ). Then, cDNA was synthesized from 500 ng of total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan), according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa; http://www.takara-bio.com). Primer pairs for amplification were as follows: for ONG1, (5’-ACTCCATCAACACCATGCTC-3’ and 5’- CGGTGCCGATCGCCGAGTGA-3’); for ONG2&3, (5’-GGCCATTAGCATCGCAAGCT-3’ and 5’-CCTCATCCACCAGGAATGCC-3’); and for ONG4, (5’-GGATCGACGAGGTGAGGAGG-3’ and 5’-TCCCACCTGGTGTTCGTCAG-3’). Ubiquitin (Os06g0681400) was amplified as an internal reference using the primer set (5’-GTGGTGGCCAGTAAGTCCTC-3’ and 5’-GGACACAATGATTAGGGATCA-3’).
Application of RiceXpro
To investigate expression levels of the HMW rice allergens in endosperm and embryo (Additional file 1: Figure S1), we utilized the low expression-level data of reproductive organs (inflorescence, anther, pistil, ovary, embryo, and endosperm) of RiceXpro (Sato et al., ). The signal intensity was normalized by the 75th percentile. The signal intensity represented in the bar graphs shows the average of three biological replicates ± SD. The signal intensity of each gene was calculated as follows: (Row signal intensity/75th percentile of each array) × 2,725. The average of the 75th percentiles from all of the arrays was 2,725.
We thank Ms. Y. Ikemoto, K. Miyashita, Y. Suzuki, M. Utsuno, and Y. Yajima for technical assistance. This work was supported by research grant from the Ministry of Agriculture, Forest, and Fisheries of Japan (Genomics and Agricultural Innovation no. GMC0006 to F.T and T.M.).
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