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, 7 (10), e48287

Genetic Modification of the Soybean to Enhance the β-Carotene Content Through Seed-Specific Expression

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Genetic Modification of the Soybean to Enhance the β-Carotene Content Through Seed-Specific Expression

Mi-Jin Kim et al. PLoS One.

Abstract

The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T(2) generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic representation of the binary vectors used in the soybean transformations.
Two vectors contained the same recombinant PAC gene as a closed box downstream of a soybean seed specific β-conglycinin promoter and a cauliflower mosaic virus 35S promoter, respectively. The remaining construct components had the same configuration, consisting of the Bar cassette to express the DL-phosphinothricin resistance gene driven by the 35S promoter and the cauliflower mosaic virus 35S terminator. LB and RB, left and right borders for Agrobacterium-mediated transformation, respectively; 5′-β, a soybean seed specific β-conglycinin promoter; 5′-35S, a cauliflower mosaic virus 35S promoter; PAC, a recombinant Psy-2A-Tp-CrtI gene; T35S, a cauliflower mosaic virus 35S terminator.
Figure 2
Figure 2. Agrobacterium-mediated transformation of half-seed explants and regeneration.
(a) Half seed explants immediately after infection (left) and at five days after inoculation (right). (b) Shoot induction medium without DL-phosphinothricin. (c) Shoot induction medium containing DL-phosphinothricin (10 mg/L) for Basta® selection. (d) Shoot elongation medium with DL-phosphinothricin (5 mg/L). (e) Rooting medium. (f) Acclimated putative transgenic plant in a small pot. (g) Transgenic plant growing in a large pot. (h) Leaf painting assay showing wild type plant sensitivity (left) and transgenic plant resistance (right) to Basta® at five days after treatment with DL-phosphinothricin (100 mg/L).
Figure 3
Figure 3. Photographs of β-PAC and 35S-PAC transgenic soybean seeds.
(a) T1 seeds. (b) T2 seeds. (c) Cross sections of T2 seeds. NT, non-transgenic plants; EV, empty vector-transgenic plants.
Figure 4
Figure 4. Determination of insertion events of β-PAC and 35S-PAC transgenes.
(a) Genomic Southern blot analysis was performed with genomic DNAs from each leaf tissues and a PAC probe. The DNA molecular size markers are indicated on the left. (b) Quantitative real-time PCR analysis was carried out with the same genomic DNAs and a Bar primer set. Con, transgenic plant that its single Bar gene insertion was already confirmed NT, non-transgenic plants; EV, empty vector-transgenic plants.
Figure 5
Figure 5. Reverse-transcriptase PCR (upper) and quantitative real-time (bottom) analyses of β-PAC and 35S-PAC transgenic soybean lines.
(a) Seeds. (b) Leaves. NT, non-transgenic plants; EV, empty vector transgenic plants. The 7, 13, 16 and 22 β-PAC transgenic lines and 5 and 6 35S-PAC transgenic lines were analyzed at the T2 generation. The Actin11 gene was used as the normalization control for both the seed and leaf RNA levels.
Figure 6
Figure 6. Total carotenoid content and β/α ratio analysis of β-PAC and 35S-PAC transgenic soybean plants.
(a) Seeds. (b) Leaves. Total carotenoid levels were calculated as the sum of eight carotenoid subtype levels i.e. violaxanthin, antheraxanthin, lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin, α-carotene and β-carotene. The β-carotenoids include violaxanthin, antheraxanthin, zeaxanthin, β-cryptoxanthin and β-carotene and α-carotenoids include lutein and α-carotene in the determination of the β- to α- carotenoid ratios. Values (µg/g dry weight) are the means of three replicates. Error bars represent the standard deviations.
Figure 7
Figure 7. Carotenoid composition of β-PAC and 35S-PAC transgenic soybean plants and their HPLC chromatograms.
(a) Seeds. (b) Leaves. The composition of individual carotenoids is shown for representative lines for β-PAC (line 16) and 35S-PAC (line 6). More detailed results of carotenoid composition analyses are listed in Table S1. (c) Representative HPLC chromatograms of transgenic seeds from β-PAC and 35S-PAC lines and transgenic leaves of β-PAC lines. Carotenoid standards included violaxanthin (vio), antheraxanthin (ant), lutein (lut), zeaxanthin (zea), α-cryptoxanthin (α-crypt), β-cryptoxanthin (β-crypt), α-carotene (α-car) and β-carotene (β-car). Values (µg/g dry weight) are the mean of three replicates. Error bars represent the standard deviations.
Figure 8
Figure 8. Tocopherol and phytosterol composition in the seeds of β-PAC and 35S-PAC transgenic soybean plants.
(a) Tocopherols. (b) phytosterols. The individual compositions in representative lines for β-PAC (line 16) and 35S-PAC (line 6) are shown. Values (µg/g dry weight) are the mean of three replicates. Error bars represent the standard deviations.

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Grant support

This work was supported by the Rural Development Administration (Code PJ006834 to SHH) and a grant from the Next-Generation BioGreen 21 Program (Code PJ008184 to SHH and PJ007978 to YSC), Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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