Seed-Specific Expression of Apolipoprotein A-IMilano Dimer in Engineered Rice Lines

doi:10.1016/j.rsci.2023.09.001

Abstract

Abstract:

Apolipoprotein A-I_Milano (ApoA-I_M) has been shown to significantly reduce coronary atherosclerotic plaques. However, the preparation of cost-effective pharmaceutical formulations of ApoA-I_M is limited by the high cost and difficulty of purifying the protein and producing the highly effective dimeric form. The aim of this study was to create an expression cassette that specifically drives the expression of dimeric ApoA-I_M in the protein bodies of rice seeds. The ApoA-I_M protein under control of the 13 kDa prolamin promoter is expressed exclusively in its dimeric form within the seeds, and immunocytochemical and immunogold analyses confirmed its expression in different caryopsis tissue such as seed coat, aleurone cell and endosperm, particularly in amyloplast and storage vacuoles. A plant-based ApoA-I_M production system offered numerous advantages over current production systems, including the direct production of the most therapeutically effective dimeric ApoA-I_M forms, long-term protein storage in seeds, and ease of protein production by simply growing plants. Therefore, seeds had the potential to serve as a cost-effective source of therapeutic ApoA-I_M.

Key words: apolipoprotein A-I_Milano, engineered plant, immunofluorescence, immunogold analysis, rice, seed-specific promoter

Serena Reggi, Elisabetta Onelli, Alessandra Moscatelli, Nadia Stroppa, Matteo Dell’Anno, Kiril Perfanov, Luciana Rossi. Seed-Specific Expression of Apolipoprotein A-I_Milano Dimer in Engineered Rice Lines[J]. Rice Science, 2023, 30(6): 587-597.

Figures/Tables 4

Fig. 1. Western blot under reducing and non-reducing conditions, and the model of ApoA-I dimer by Gogonea et al (2013). A, Western blot under reducing conditions (with β-mercaptoethanol); C+, ApoA-I recombinant; C-, Untransformed rice; ID 15, 20, 23, and 24 rice plants were positive in the screening PCR. All plants showed a band at 28 kDa, similar to the positive control, which was not present in the wild type (WT, untransformed rice) seeds. B, Relative expression of ApoA-IMilano gene in rice plants. RNA was extracted from rice seeds of different plants, and beta-tubulin was used as housekeeping gene. ID 15, 20, 23, and 24 rice plants were positive in the screening PCR. Results are presented as Mean ± SE from three biological replicates. Different lowercase letters above the bars indicate statistically significant differences at the level of P ≤ 0.05.

Fig. 2. Temporal and spatial expression of ApoA-IM gene. A, Western blot under reducing conditions (with β-mercaptoethanol). Rice seeds from the same panicle were collected at different days after flowering. Lines 1, 2, 3, 4, 5, 6, and 7 represent 4, 8, 12, 16, 20, and 25 days after flowering, and maturity stage, respectively. B, qRT-PCR analysis of ApoA-IM gene expression levels in seeds at different ripening stages. Results are presented as Mean ± SE from three biological replicates. Different lowercase letters above the bars indicate statistically significant differences at the level of P ≤ 0.05. C, Western blot under reducing conditions was performed on different plant tissues of the same transformed rice plant. The presence of the signal corresponding to positive control confirmed the seed-specificity of 13 kDa promoter. D, RT-PCR carried out on different plant tissues. Beta-tubulin was used as the internal control. C+, Recombinant hApoA-I; L, Proteins extracted from the leaf; S, Proteins extracted from transformed rice seeds; Cu, Proteins extracted from culm; R, Proteins extracted from roots; C-, Rice seeds from the untransformed plant.

Fig. 3. Immunofluorescence and bright field images of caryopses I (A-F) and II (G-N) of ApoA-IM transformed plants. A-F, Immunofluorescence (A, C, and E) and bright field images (B, D, and F) of caryopsis I of ApoA-IM transformed plant. For A and C, a high fluorescence was observed only in the amyloplasts of endosperm cells particularly in the stroma, surrounding starch granules in C with arrows. E and F, Negative controls showed no cross-reaction with seed tissues. G-N, Immunofluorescence (G, I, K, and M) and bright field images (H, J, L, and N) of caryopsis II of ApoA-IM transformed plant. Pericarp and nucellus appeared thinner in G, H, I, J, M, and N. ApoA-IM was localized in organelles in endosperm cells, seed coats, and aleurone cells in I and K with arrows. G and I, High fluorescence was observed in the seed coat cells. M and N, Only autofluorescence was observed in negative control. a, Aleurons cell; n, Nucellus; Pr, Pericarp; sc, Seed coat; se, Seed endosperm cell.

Fig. 4. Immunogold analysis on caryopsis II of ApoA-IM transformed plants. A and B, ApoA-IM was localized into plasmids in the chloroplast grana in the seed coat (B is a magnification of box in A). C, Dark spots were observed in the primary cell wall and not in middle lamellae (with arrows). D, In aleuron cells, ApoA-IM was localized in storage vacuoles (with arrows). E, ApoA-IM was localized into plasmids in amyloplasts of the endosperm cells (with arrows). F, In aleuron cells, ApoA-IM was localized in vesicles associated with dictyiosomes (with arrowhead). cl, Chloroplast; cw, Cell wall; ml, Middle lamellae; a, Amyloplast; sv, Storage vacuole; g, Golgi body.

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