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

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 T2 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.

In silico identification and analysis of phytoene synthase genes in plants

“…Overexpression of the genes related to the carotenoid biosynthesis pathway in rapeseed (Shewmaker et al, 1999), rice endosperm (Ye et al, 2000), potato tuber (Ducreux et al, 2005), and soybean (Kim et al, 2012) can significantly increase the content of carotenoids in these crops. PSY is the first rate-limiting enzyme in the synthesis process of carotenoid production and the regulation metabolic of geranylgeranyl pyrophosphate.…”

“…PSY is the first rate-limiting enzyme in the synthesis process of carotenoid production and the regulation metabolic of geranylgeranyl pyrophosphate. Previous studies demonstrated that the constitutive and specific expression of PSY could significantly improve the content of carotenoid in the tissues of higher plants, which has also become the basis of genetically modified carotenoid genes in plants (Shewmaker et al, 1999;Ye et al, 2000;Fraser et al, 2002;Ducreux et al, 2005;Kim et al, 2012).…”

Unraveling the light-specific metabolic and regulatory signatures of rice through combined in silico modeling and multi-omics analysis

“…Carotenoids were extracted and measured using HPLC as described previously (Kim et al, 2012a). Briefly, the carotenoids were extracted from rice samples (0.12 g) by adding 3 mL of ethanol containing 0.1% (w/v) ascorbic acid, vortex mixing for 20 s, and placing in a water bath at 85°C for 5 min.…”

A novel phytoene synthase paralog from halophytic Salicorniaconfers salinity tolerance in plants

“…Golden rice is a glaring example of this promotion (Ye et al, 2000;Paine et al, 2005). Other metabolically manipulated plants for carotenoid improvement are tomato (Fray et al, 1995) canola (Shewmaker et al, 1999), wheat (Cong et al, 2009), potato (Romer and Fraser, 2005), and soybean (Kim et al, 2012). However, knowledge about the role of genes involved in carotenoids biosynthesis for induction of salt tolerance and their expression analyses remained obscure.…”