Fig 1.
Transgenic rice seed used in the study.
(a) Four gene cassettes expressed in transgenic rice seeds. GluB4 P, glutelin B4 promoter; 16 kD P, 16 kDa prolamin promoter; 10 kD P, 10 kDa prolamin promoter; GluB1 P, Glutelin B1 promoter; mGluA, modified glutelin A2 coding region; mGluB, modified glutelin B1 coding region; mGluC, modified glutelin C coding region; GluB4 T, glutelin B4 terminator; 16 kD T, 16 kDa prolamin terminator; 10 kD T, 10 kDa prolamin terminator; GluB1 T, glutelin B1 terminator; F1, Cry j 1 F1; F2, Cry j 1 F1; F3, Cry j 1 F3; shCry j 2, shuffled Cry j 2; SP, Glutelin B1 signal peptide; KDEL, ER retention signal. (b) SDS-PAGE (CBB) and immunoblot (IB) analyses of transgenic rice seed (T5–T7). The positions of glutelin precursor, glutelin acidic or basic, globulin, and prolamin on the SDS-PAGE gel are indicated on the right side of the panel. Immunoblot detection of transgene products derived from four gene cassettes.
Fig 2.
Schematic of the preparation of concentrated PB product.
(a) Outline describing the preparation of concentrated PB product. (b) SDS-PAGE and immunoblot analyses of brown rice, bran, milled rice, and concentrated PB product in wild-type and transgenic rice seed. The amounts of seed proteins in the concentrated PB product were approximately 12-fold higher than those in milled rice; therefore, total protein extracts of concentrated PB products were diluted before SDS-PAGE.
Table 1.
Component analysis of milled rice powder and concentrated PB product
Fig 3.
Detection of residual α-amylase in the concentrated PB product.
(a) One-microliter of protein extract from 2 mg of concentrated PB product was subjected to immunoblot analysis (five independently prepared samples). The α-amylase level in each signal was estimated by comparison with standards (2.5, 5.0, 10, and 25 ng) of α-amylase purified from α-amylase Termamyl120L. The amount of residual α-amylase was calculated as a percentage. (b) Detection of residual α-amylase during the preparation of concentrated PB product. Supernatants obtained in the first centrifugation after α-amylase treatment (lane 1), second to fourth centrifugation after washing (lane 2 to lane 4) and protein extracts from the final pellet (concentrated PB product, lane 4) were analyzed by SDS-PAGE (CBB) and immunoblotting (IB). A volume of 2 μL of sample was subjected to SDS-PAGE. Experiments were repeated two times (Exp 1 and Exp 2).
Fig 4.
Evaluation of shelf life of the concentrated PB product.
Concentrated PB products were prepared from transgenic rice seeds and stored at room temperature for 10 month (Stocked PB). Ten month after, concentrated PB products were prepared again from the same transgenic rice seeds (Fresh PB). Total proteins were extracted from flesh and stocked concentrated PB products at the same time and subject to SDS-PAGE (CBB) and immunoblot (IB) analyses.
Fig 5.
Indirect immunohistochemical analysis of rice cells of premature seed (a–f), seed powder (g–l) and concentrated PB product (m–r).
The left panels show PB-I (red), the middle panels show PB-II (green), and the right panels show the merged images. PB-I was detected by rhodamine staining. PB-II was detected by immunohistochemical staining using an anti-GluB antibody. Low and high magnifications are shown. Arrowheads indicate PB-II. Asterisks indicate starch granules.
Fig 6.
Comparison of resistance to digestive enzyme in rice seed powder and concentrated PB product.
(a) Pepsin digestion of seed powder and concentrated PB product for 0, 1, 5, 15, 30, 60, and 120 min at 37°C. Samples consisted of 10 mg of seed powder or 1 mg of concentrated PB products. (b) Pepsin digestion of recombinant protein after extraction from transgenic rice seeds for 0, 0.5, 1, 3, 5, 15, 30, and 60 min.
Fig 7.
Stepwise extraction of seed proteins from seed powder and concentrated PB product.
(a) Flow chart of stepwise extraction. (b) SDS-PAGE (CBB) and immunoblot (IB) analyses are shown. Lane 1, globulin fraction; Lane 2, endogenous glutelin fraction; Lane 3, prolamin fraction; Lane 4, recombinant protein fraction; and Lane 5, pellet fraction.
Fig 8.
Uptake of antigen (GFP-fused shuffled Cry j 2) by the intestinal tract.
(a) Ileum tissues before oral administration of rice seed powder expressing GFP-fused shuffled Cry j 2. (b), (c) and (d) Ileum tissues at 4, 8, and 12 h after oral administration of rice seed powder expressing GFP-fused antigen. (e) Ileum tissues at 12 h after oral administration of non-transgenic rice. (f) Jejunum tissues at 12 h after oral administration of rice seed powder expressing GFP-fused antigen. (g) Average number of GFP spots in 20 regions of a 100 μm2 area of ileum and jejunum tissue sections 12 h after oral administration of rice seed powder. WT, wild type; Tg, transgenic rice.
Fig 9.
Oral administration of rice seed powder and concentrated PB product.
(a) The levels of serum allergen-specific IgE were examined by ELISA. (b) Allergen-specific splenic CD4+ T-cell proliferative responses were expressed as stimulation index. Data are expressed as the mean ± standard deviation (n = 3 mice per group). ** P < 0.01 and * P < 0.05 for the group of mice fed Tg-rice seed powder or Tg-PB product in comparison with the group of mice fed WT-rice seed powder or WT-PB product, respectively. WT-rice, wild type rice; Tg-rice, transgenic rice; WT-PB, wild-type concentrated PB; Tg-PB, transgenic concentrated PB.