Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds

“…Also, as shown in Fig. 6, the yellow color in Glb::stPAC seeds [32] was largely intensified to an orange color in the PGD1::OsDXS2_Glb::stPAC lines, but remained just yellow in the PGD1::OsDXR_Glb:: Chlorophyll absorbance in the same leaves as in a measured by spectrophotometry. NT represents non-transgenic rice plants (Oryza sativa L. cv.…”

“…a Built-in pathway for carotenoid biosynthesis in rice plants; CRTI, Pantoea annatis desaturase; DMAPP, dimethylallyl diphosphate; DXP, deoxyxylulose 5-phosphate; DXR, DXP reductoisomerase; DXS, DXP synthase; IPP, isopentenyl diphosphate; GGPP, geranylgeranyl pyrophosphate; G3P, glyceraldehyde 3-phosphate; LCYB, lycopene β-cyclase; MEP, methylerythritol 4-phosphate; OsDXR, rice DXP reductoisomerase; OsDXS2, rice DXP synthase; PSY, phytoene synthase; stCrtI, rice codon-optimized synthetic gene encoding the Pantoea CrtI gene; stPsy, rice codon-optimized synthetic gene of the Capsicum gene encoding PSY; stPAC, a recombinant gene of stPsy and stCrt linked with 2A, which is the rice codon-optimized foot-and-mouth disease virus 2A peptide. b Diagrams of the four vectors for rice transformation of OsDXS2 and OsDXR with the pGlb::stPAC vector that was previously used to generate a carotenoid-intensifying trait in rice endosperm [32]. Bacterial attachment attB sites needed for Gateway cloning are marked with hatched boxes.…”

“…To introduce entire PGD1::OsDXS2::PinII or PGD1:: OsDXR::PinII cassettes into the region of the PGD1 promoter and the PinII terminator into the stPAC expression cassette, which produces β-carotene under the control of a rice endosperm-specific globulin (Glb) promoter [32], they were individually PCR-amplified with the F3/R3 primer pair and then cloned into a pGlb:: stPAC vector using the XhoI site, yielding either pPGD1:: OsDXS2_Glb::stPAC or pPGD1::OsDXR_Glb::stPAC for rice transformation (Fig. 1b).…”

“…To examine the transgene copy number in rice genomes, TaqMan real-time PCR was performed using the primer set NF/NR, a customized probe NP labeled with a 6-carboxyfluorescein dye to detect a Nos terminator on the BAR cassette (35S::Bar:: Nos), and the customized VIC dye-labeled α-tubulin probe (Os11g14220), as an internal reference (Assay ID: Os03643486_s1; Applied Biosystems, Foster City, CA). The PCR was carried out using a TaqMan Gene Expression Master Mix (Applied Biosystems), and fluorescence was measured with the CFX Connect™ Real-Time System (Bio-Rad, Richmond, CA) relative to the value of 1 copy in the homozygous T3 generation of the stPAC 25 rice line [32] using PCR conditions, as previously described [43]. The sequences of primers and probes used in the genomic DNA analysis are listed in Additional file 4: Table S7.…”

“…Carotenoids, a large family of tetraterpenoids (C 40 ), are abundant in photosynthetic and non-photosynthetic plant tissues [26], and are also essential nutrients for humans as they are the precursors of pro-vitamin A [27]. Since carotenoids are enriched in photosynthetic green parts but lacking in non-photosynthetic seed endosperms in rice plants [28,29], several "Golden Rice" varieties have been genetically engineered in which carotenoids, such as β-carotene, are enriched in endosperms [29][30][31][32] to enhance the nutritional value of rice as a staple cereal. In maize plants (Zea mays L.), the expression levels of the three maize DXS genes were examined in different organs, including the mature kernels of two varieties with white and yellow grain [13].…”