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

Plant Biotechnol Rep DOI 10.1007/s11816-017-0440-0 Online ISSN 1863-5474 Print ISSN 1863-5466 ORIGINAL ARTICLE Effect of codon optimization on the enhancement of the b-carotene contents in rice endosperm Ye Sol Jeong1 • Hyung-Keun Ku1 • Jae Kwang Kim2 • Min Kyoung You1 Sun-Hyung Lim3 • Ju-Kon Kim4 • Sun-Hwa Ha1 • Received: 21 May 2017 / Accepted: 25 May 2017 Ó Korean Society for Plant Biotechnology and Springer Japan 2017 Abstract A b-carotene is the most well-known dietary source as provitamin A carotenoids. Among b-caroteneproducing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance b-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T1 seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of bcarotene (4-fold, 2.37 lg/g) as well as total carotenoid (2.9-fold, 3.50 lg/g) relative to PAC seeds, suggesting a bbranch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production. Ye Sol Jeong and Hyung-Keun Ku contributed equally to this study. Keywords b-carotene
Codon optimization
Recombinant gene
Rice endosperm
Synthetic gene Electronic supplementary material The online version of this article (doi:10.1007/s11816-017-0440-0) contains supplementary material, which is available to authorized users. Introduction & Sun-Hwa Ha sunhwa@khu.ac.kr 1 Department of Genetic Engineering and Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 17104, Korea 2 Division of Life Sciences and Bio-Resource and Environmental Center, Incheon National University, Incheon 22012, Korea 3 National Academy of Agricultural Science, Rural Development Administration, Jeonju 54874, Korea 4 Graduate School of International Agricultural Technology and Crop Biotechnology Institute/GreenBio Science and Technology, Seoul National University, Pyeongchang 25354, Korea The development of genetically modified rice that produces b-carotene seeks to address malnutrition-associated vitamin A deficiency (VAD) problems, such as immune disorders and blindness, in developing countries (Sommer et al. 2002; Ye et al. 2000). As b-carotene is a precursor of vitamin A, consumption of b-carotene-producing rice as a staple food could be an effective way to alleviate VAD (Tang et al. 2009). By introducing two genes of phytoene synthase (Psy) and carotene desaturase (CrtI) as separated two cassettes into the rice genome, Golden Rice (GR) 1 and 2 successfully produced the carotenoids (1.6 lg/g) de novo and then largely enhanced the amounts of those (37 lg/g) up to 23 times, respectively (Paine et al. 2005; 123 Plant Biotechnol Rep Ye et al. 2000). In 2010, by introducing the single recombinant gene (PAC, Psy:2A:CrtI) as one cassette, a new type of GR (PAC rice) produced the carotenoids as a result of bicistronic expression of two genes (Ha et al. 2010). The effectiveness of the PAC gene was further proven to produce the 62-fold higher levels of total carotenoids relative to wild-type when expressed in a seedspecific manner in soybeans (Kim et al. 2012). Hence, it proved the practical use of viral 2A technology to transfer a smaller transgene into plant genomes to express Psy and CrtI simultaneously in a coordinated manner. Although this PAC gene could greatly increase the contents of total carotenoids in soybean (146 lg/g), the limited yield of carotenoids in rice endosperm (1.30 lg/g) has been a barrier to practical use as biofortified rice plants (Ha et al. 2010; Kim et al. 2012). In plants, metabolic engineering for specialized metabolites and molecular farming for high-value proteins with industrial importance require a sufficient quantity of protein production through the efficient expression of transgenes. In these fields of plant biotechnology, codon modification has been widely used to optimize the expression of heterologous transgenes to match the codon usage of the host plants, including rice and tobacco plants (Al-Babili et al. 2006; Kim et al. 2016; Li et al. 2016; Liu et al. 2011; Misztal et al. 2004). Each living organism has a preferred codon usage and its codon bias plays an important role as a determinant of the translation efficiency as the subset of optimal codons corresponds to the most abundant tRNAs (Ikemura 1981; Kanaya et al. 1999). Thus, one of the typical approaches for codon optimization is to replace rare codons in a transgene into abundant ones of the host to elevate translational efficiency (Gustafsson et al. 2004; Tuller et al. 2010). In addition, codon optimization also matches the GC content of nucleotides suitable for the host and alters unstable RNA sequences derived from different species, thus increasing both transcriptional efficiency and transcript stability (Jackson et al. 2014; Li et al. 2015). In this study, we adopted a codon optimization strategy by altering the heterologous codons of transgenes to ricepreferred ones to increase gene expression and to improve b-carotene content in rice endosperm. The DNA sequences of two original genes, Capsicum Psy (Psy) and Pantoea CrtI (CrtI), constituting the PAC recombinant gene, were artificially modified based on a rice codon usage into the synthetic PAC gene (stPAC, stPsy:2A:stCrtI) including the synthetic Psy (stPsy) and the synthetic CrtI (stCrtI) gene. The effectiveness of codon optimization in stPAC rice in comparison to PAC rice was evaluated by analyzing transgene expression, color phenotype, and carotenoid content in transgenic rice endosperm. 123 Materials and methods Codon optimization and gene synthesis Based on the native PSY protein sequence (419 aa) from Korean red pepper (Capsicum annuum), and CRTI (493 aa) from Pantoea ananatis (formerly Erwinia uredovora), the nucleotide sequences of Psy and CrtI were modified to derive the optimized sequences of stPsy and stCrtI with referred codon usage for Oryza sativa using Leto software (Entelechon, Regensberg, Germany) and a rice codon usage table (http://www.kazusa.or.jp/codon/). The 2A peptide sequence for bicistronic expression of two genes and a transit peptide sequence (Tp) for chloroplast targeting of CRTI were derived from a previously used PAC gene (Ha et al. 2010). The final DNA sequence of stPAC (2,952 bp), which was a sequential assembly of stPsy, 2A, Tp, and stCrtI, was artificially synthesized using a customized service (Entelechon). To facilitate the Gateway cloning procedure, the stPAC gene was inserted into the pCR4-TOPO vector with attL1 and attL2 (Invitrogen, Waltham, MA). Vector construction and rice transformation For rice transformation, the stPAC gene in the pCR4-TOPO vector was directly recombined into the pMJ103 destination vector with an endosperm-specific rice globulin promoter (Myungji University, Yongin, Korea) using a Gateway cloning strategy (Karimi et al. 2002), yielding the pstPAC construct (Fig. 1). Gateway cloning was performed using GatewayÒ LR ClonaseÒ II Enzyme Mix in accordance with the manufacturer’s instructions (Invitrogen). The pstPAC in Escherichia coli strain (DH5a) was introduced into an Agrobacterium tumefaciens strain (LBA4404 harboring the pSB1 vector) by tri-parental mating with a conjugated helper strain (HB101 harboring the pRK2013 plasmid). Embryogenic calli that proliferated from the mature seeds of japonica-type Korean rice (Oryza sativa L., cv. Hwayoung) were co-cultivated with a prepared Agrobacterium strain possessing the pstPAC vector and regenerated into transgenic rice plants via shooting and rooting procedures on selection medium containing phosphinothricin (4 mg/L) and cefotaxime (500 mg/L) (Park et al. 2015). Growth conditions and visual inspection of seed color Putative transgenic plants (T0) were acclimatized in a cultivation room and grown in a greenhouse at 28 °C until maturity. Seeds of transgenic plants were harvested Plant Biotechnol Rep Fig. 1 Schematic representation of the PAC and stPAC constructs for rice transformation. Each pPAC and pstPAC vector contains the recombinant gene, Psy:2A:CrtI (PAC) and stPsy:2A:stCrtI (stPAC), respectively. The remaining components in each vector construct are exactly the same and include the endosperm-specific rice globulin promoter (P-Glb), and the 30 region of the potato proteinase inhibitor II gene (T-PinII). The Bar in the big arrow represents a cassette used to express the Bar gene under a 35S promoter (P-35S) with a 30 region from the nopaline synthase gene (T-Nos). The primer positions used for qRT-PCR (used in Figs. 4a, 5 and listed in Table S1) to detect transcripts of 2A-Tp (2A-F/Tp-R), stPsy (stPsy-F/stPsy-R), and stCrtI (stCrtI-F/stCrtI-R) are indicated. The 50 -matrix attachment region (Mar) from the chicken lysozyme gene flanked at both ends of T-DNA was used as a probe in Southern blot analyses. BR right border, BL left border, 2A rice codon-optimized DNA sequence encoding 2A peptide from foot-and-mouth disease virus, Tp transit peptide of rice rubisco small subunit, RI EcoRI restriction enzyme sites 40 days after flowering (DAF) in the green house (T1 generation) and in the field (T2 and T3 generation). To harvest PAC and stPAC seeds at the same generation (T3), T2 plants from PAC line 4 and stPAC line 21, 25, 102 were grown in the same field. The seed colors of the nontransgenic and transgenic rice plants were visually inspected after dehusking (TR-200 Electromotion rice husker; Kett, Tokyo, Japan) and polishing (Pearlest polisher; Kett). (Nos-M) labeled with the fluorescent reporter dye 6-carboxy-fluoroscein (6-FAM) to detect the Nos terminator in a Bar gene expression cassette in accordance with the manufacturer’s protocols on a CFX ConnectTM Real-Time System (Bio-Rad, Richmond, CA). An a-tubulin assay (Assay ID: Os03643486_s1; Applied Biosystems) using the fluorescent reporter dye VIC was performed as an internal control (Song et al. 2016). The PCR reaction conditions were set as follows: 10 min enzyme activation at 95 °C, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C. Data were analyzed to calculate the T-DNA copy number using Bio-Rad CFX manager software (Bio-Rad) using the homozygous transgenic rice line, PAC 4, which was assigned a value of 1 as a single-copy reference (Ha et al. 2010). The sequences of primers for genomic DNA analysis are provided in the supporting information (Table S1). Genomic DNA analysis Using a modified cetyltriammonium bromide-based protocol (Allen et al. 2006), young leaf tissues from the T0 generation were used to extract genomic DNAs to estimate the copy number of the transgene in rice plants. Southern blot analysis was performed using five micrograms of DNA digested with EcoRI, fractionated in a 1% agarose gel, and transferred onto a Hybond-N? nylon membrane (Amersham Biosciences, Piscataway, NJ). The membrane was hybridized with a Mar gene probe that was generated with Mar-F/Mar-R using a PCR DIG Probe Synthesis Kit (Roche Inc., Mannheim, Germany), and signals were developed using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) according to the manufacturer’s instructions. The membrane was exposed to Lumi-Film Chemiluminescent Detection film (Roche) and the bands were imaged with LAS 4000 software (Fujifilm, Tokyo, Japan). Taqman PCR analysis was carried out with Taqman Gene Expression Master Mix (Applied Biosystems, Foster City, CA) using a primer set (Nos-F/Nos-R) and a probe RNA analysis Total RNA was extracted from 100 mg of mature seeds at 40 DAF after imbibition in water and trituration in a mortar with liquid nitrogen using PureLinkÒ Plant RNA Reagent (Invitrogen) according to the manufacturer’s instructions. The RNAs were treated with DNase I (Qiagen, Hilden, Germany) to remove any remaining genomic DNA and their quality and concentration were checked via agarose gel electrophoresis and a NanoDrop Spectrophotometer ND-2000 (Nano-Drop Technologies, Wilmington, DE). cDNAs were simultaneously synthesized and amplified from 1 lg of total RNA using AccuPowerÒ RT Premix (Bioneer, Daejeon, Korea) according to manufacturer’s instructions. Quantitative real-time (qRT) PCR was carried 123 Plant Biotechnol Rep out using a CFX ConnectTM Real-Time System (Bio-Rad). Each reaction was composed of 100 ng of cDNA, 0.6 lM of gene-specific primers, 10 lL SYBR Green Real-time PCR master mix (Bio-Rad) in a total volume of 20 lL. The PCR conditions were as follows: 3 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 30 s at 60 °C. The primers used to amplify target genes were as follows: F2A-RTp (2A-F/Tp-R), stPsy (stPsy-F/stPsy-R), and stCrtI (stCrtI-F/ stCrtI-R). The rice ubiquitin 5 gene (OsUbi5, Os01g22490) was used as an internal control using two OsUbi5 primer sets (OsUbi-QA/OsUbi-QR, and OsUbi-QF/OsUbi-QR) (Jain et al. 2006). Each qRT-PCR reaction was carried out in triplicate on three individual plants. Information on the primers used for qRT-PCR is listed in the supporting information (Table S1). Protein analysis Protein from 100 mg of rice seed powder, prepared by soaking rice in water and grinding it under liquid nitrogen, was extracted using buffer containing 0.025 M Tris–HCl (pH 6.8), 4% SDS, 4 M Urea, and 5% 2-mercaptoethanol. The supernatants were collected from extracts after centrifugation at 12,0009g for 45 min at 4 °C, and protein concentrations were measured using a Quant-iTTM Protein Assay Kit (Invitrogen) according to the manufacture’s protocol. A total of 80 lg of protein was separated by electrophoresis on an 8% SDS/polyacrylamide gel and transferred onto a PVDF membrane (Whatman, Kent, UK) using a Trans-Blot SD semi-Dry electrophoretic transfer cell (Bio-Rad). Western blot analyses were performed using primary anti-CRTI antibody (1:2500) raised against the Pantoea CRTI protein (kindly provided by Dr. Peter Beyer group at University of Freiburg in Germany). Bound antibody was detected using an anti-rabbit IgG (Fc) alkaline phosphatase-conjugated secondary antibody (1:5000) and a BCIP/NBT color development system (Promega, Madison, WI). The bands were imaged using LAS 4000 software (Fujifilm) and densitometric analysis was carried out using Image QuantTL software (GE Healthcare, Piscataway, NJ). Carotenoid extraction and quantitation Mature seeds at 40 DAF were dehusked after harvesting and drying and were ground into a fine powder using a pestle for the extraction and quantitation of carotenoids by high-performance liquid chromatography (HPLC). In brief, 1.0 g of the homogenized sample was prepared for carotenoid extraction with 3 mL of ethanol containing 0.1% ascorbic acid (w/v) and saponified with potassium hydroxide (120 lL, 80%, w/v). After adding b-Apo-80 carotenal (0.1 mL of 25 lg/mL) as an internal standard, the 123 carotenoids were extracted twice with 1.5 mL of hexane followed by centrifugation at 1,2009g for layer separation. Nitrogen-dried extract aliquots were re-dissolved in 50:50 (v/v) dichloromethane/methanol before HPLC analysis. Carotenoid separation was performed on an YMC ODS C-30 column (3 lm, 4.6 mm 9 250 mm; YMC Europe, Germany) using an HPLC system (Agilent 1100 Series; Agilent, Santa Clara, CA). Chromatograms were generated at 472 and 450 nm for lycopene and the other four compounds of a-carotene, b-carotene, lutein, and zeaxanthin, respectively. All details, solvent information and elution conditions, were as described previously (Kim et al. 2010). Carotenoid standards for lycopene (w,w-carotene) a-carotene (b,e-carotene), 13Z-b-carotene, (all-E)-b-carotene, 9Z-b-carotene, lutein (b,e-carotene-diol), and zeaxanthin (b,b-carotene-diol) were purchased from CaroteNature (Lupsingen, Switzerland). The amount of b-carotene (b,bcarotene) was calculated as the sum of 13Z-b-carotene, (all-E)-b-carotene, and 9Z-b-carotene. With respect to peak area ratios of the analyte to internal standard, calibration curves were drawn by plotting different concentrations of each carotenoid standard. Results Codon-optimization and artificial synthesis of the stPAC gene based on the PAC gene for rice To enhance the levels of carotenoids via gene modification in rice endosperm, the DNA sequences of two carotenoid biosynthetic genes originating from heterologous sources, C. annum for Psy and Pantoea ananatis for CrtI, were optimized for rice plants using the O. sativa codon usage database (http://www.kazusa.or.jp/codon/) (Nakamura et al. 2000). To minimize codon bias, codons less frequently used were replaced with more frequently used ones based on the same protein sequences of the PAC recombinant gene. The codon adaptation index (CAI) calculated using a CAI calculator (http://ppuigbo.me/programs/CAI cal/) (Puigbo et al. 2008) resulted in codon alterations of 64.0% (268/419 aa) and 65.1% (321/493 aa) and DNA similarities of 74.1 and 75.3% between original and modified genes encoding PSY and CRTI, respectively. Additionally, the CAIs of the two modified genes encoding PSY and CRTI were increased to 0.84 and 0.85 from 0.73 and 0.7, while the GC contents were also increased to 57.8 and 57.9% from the 45.2 and 53.1% found in the original genes, respectively. These percentages are similar to the average GC content of O. sativa genes (about 55%) (Li et al. 2015). A new version of a recombinant gene, the stPAC gene, including two codon-optimized genes linked with the same 2A and Tp sequences derived from the Plant Biotechnol Rep original PAC recombinant gene, was artificially synthesized. Consequently, the overall GC content and CAI of the stPAC gene were increased from 50.9 to 58.7% and from 0.77 to 0.85, respectively, compared to the PAC gene. The stPAC gene consisting of a single open-reading frame for the expression of two genes was specifically expressed in rice endosperm under the control of the rice globulin promoter using the same rice transformation vector previously used to express the PAC gene (Ha et al. 2010). The regions between the left and right T-DNA borders in the corresponding pPAC and pstPAC vectors are depicted schematically in Fig. 1. Effect of copy number of a stPAC transgene on carotenoid content in rice endosperm Using co-cultivation with an Agrobacterium strain harboring the pstPAC gene, a total of 67 stPAC transgenic rice lines were obtained and the copy number of T-DNA was analyzed by Southern blotting and Taqman PCR analyses using leaf tissues from the T0 generation. The copy numbers in these stPAC lines were one (24 lines), two (24 lines), three (8 lines), and more than four (11 lines). Their T1 seeds were harvested and the yellow phenotype was observed and compared to the white phenotype of nontransgenic rice seeds. Additionally, HPLC analysis revealed the levels of carotenoid resulting from de novo synthesis from the introduced stPAC gene in the transgenic rice endosperm. An average of 2.43 lg/g of total carotenoids was measured, with a range of a minimum of 0.43 lg/g and a maximum of 4.45 lg/g (Fig. 2). The average content of carotenoids was enhanced as the copy number of the transgene increased, resulting in 2.17 (one Fig. 2 Levels of carotenoid content in T1 stPAC seeds. Total amounts of carotenoid in 67 stPAC seeds carrying one (24 lines), two (24 lines), three (8 lines), and more than four copies (11 lines) of the stPAC gene were determined by HPLC analysis at the T1 generation. Individual and average carotenoid contents of stPAC endosperm with their corresponding copy numbers are indicated by an open circle (s) and dash (–), respectively copy), 2.32 (two copies), and 2.93 lg/g (three copies) of total carotenoids. Interestingly, insertion of over four genes (an average 2.91 lg/g of four to nine copies) resulted in no additional gene dosage effect. This implies a positive correlation between carotenoid contents resulting from stPAC gene expression and the copy number of the stPAC gene up to three in rice endosperm, suggesting that transgenes over three could have more chances to cause negative effects on insertional position. Also, it suggests that the good performance of carotenoid production could be generated in single-copy transgenic plants if sufficient numbers of transformants were obtained and analyzed for the target trait. Effect on protein expression from the stPAC transgene in rice endosperm Representative lines with the highest levels of carotenoid were selected from among the 67 transgenic plants based on the copy number of T-DNA integration (Fig. 2). Specifically, three lines with one copy (No. 21, 25, 102), three lines with two copies (No. 78, 85, 88), four lines with three copies (No. 6, 8, 49, 73), and five lines with over four copies (18, 95, 99, 117, 123) were selected. Their T1 seeds were analyzed by Western blotting with an anti-CRTI antibody to detect the downstream protein of the bicistronic construct in stPAC, as well as PAC rice plant (Fig. 3). The levels of expression of the CRTI protein in 15 stPAC plants were compared relative to a T5 seed of the PAC line 4 that was assigned a value of one in a previous study, which confirmed equivalent levels of CRTI expression among four PAC lines with a single copy transgene (Ha et al. 2010). All transgenic rice lines for PAC and stPAC displayed two signal bands for CRTI including a CRTI protein of 55 kDa (cleaved protein) and a fused PSY:2A:CRTI of 110 kDa (uncleaved protein product), in addition to a nonspecific product (NSP) band around 60 kDa was a crossreacting product in all samples including non-transgenic (NT) rice (Fig. 3). The stPAC lines exhibited variable levels of CRTI protein expression, but most of them, except for stPAC line 78, showed higher levels of expression than the PAC line 4. The average fold intensities of CRTI expression in the stPAC endosperm were increased to 3.3 (3, 4, and 3 in lines 21, 25, 102 with one copy), 3.7 (1, 5, and 5 in lines 78, 85, 88 with two copies), 6.3 (8, 8, 3, and 6 in lines 6, 8, 49, 73 with three copies), and 5.8 (3, 7, 6, 8, and 5 in lines 18, 95, 99, 117, 123 with more than four copies) relative to a value of 1 in PAC 4 endosperm (Fig. 3). These results confirmed that codon-optimization of stPAC from PAC for rice reliably enhances expression of protein in endosperm. Also, the higher expression of protein may be caused by the integration of more than a single copy of the stPAC, as demonstrated by the positive 123 Plant Biotechnol Rep Fig. 3 Levels of CRTI protein in T1 stPAC seeds. Western blot analysis was performed using anti-CRTI antibody raised against Pantoea CRTI protein in 15 stPAC seeds chosen from among 67 lines at the same T1 generation based on high carotenoid contents. Uncleaved (PSY:2A:CRTI) and cleaved (CRTI) products are indicated by solid arrows and a non-specific product (NSP) is indicated by dotted arrows. NSP was used as a loading control to determine band intensities calculated using Image QuantTL software. Intensity fold was calculated using CRTI band of PAC 4 line as a reference 1. NT designates non-transgenic plants used as a negative control correlation between expression levels and gene copies up to three copies demonstrated in Fig. 2. Furthermore, to compare the effectiveness of codon optimization of stPAC from the PAC gene, three lines with a single copy of the transgene showing stable levels of protein production were selected and further developed to a homozygous generation. around 60 kDa, was observed. stPAC 21, 25, and 102 displayed 3.0-, 2.5-, and 2.9-fold greater intensities than that observed in PAC 4 line when normalized using the NSP signal. This indicates that the translation efficiency of the modified stPAC gene was significantly improved on average by a factor of 2.8-fold relative to the original PAC gene (Fig. 4b). Despite these similar levels of CRTI protein among the three stPAC lines, different levels of the 2A-Tp transcripts were examined in the regions of stPsy and stCrtI by qRTPCR (Fig. 5a, b). Both amounts of stPsy and stCrtI transcripts were resulted in the same pattern of the highest expression in stPAC line 102 and relatively lower expression in stPAC 21 and 25 lines as detected with that of 2ATp (Fig. 4a). Hence, these results demonstrate that the codon optimization might enhance translation efficiency as estimated by quantification of Western bands (Fig. 4b) irrespective of diverse transcript levels (Figs. 4a, 5a, b). It still implies that the codon optimization can enhance the stability of transcripts. Comparison of transgene expression in the codonoptimized stPAC relative to PAC transgenic rice endosperm To define the effect on transgene expression resulting from codon optimization of stPAC based on the PAC gene, the stPAC 21, 25 and 102 lines with single intact copies, as confirmed by Taqman PCR (Fig. S1a) and Southern blot (Fig. S1b) analyses, were selected since high carotenoid content and stable protein expression among these lines had been analyzed (Figs. 2, 3). To ensure the comparability of stPAC relative to PAC lines, they were grown in the same field during the same year as PAC 4 with the single copy of the transgene as a reference line (Ha et al. 2010). Additionally, their seed ages were also matched to a coeval generation at T3 by transplanting the same T2 plants. The transcriptional efficiencies of the PAC and stPAC recombinant genes were first compared by qRT-PCR using identical nucleotide sequences as primers in the 2A and Tp region (Fig. 4a). Transcript levels of 2A-Tp were 1.4- and 2.8-fold higher in stPAC lines 25 and 102, but were similar or decreased by 0.9-fold in stPAC line 21, than transcript levels in the PAC 4 line. This suggests that transcriptional efficiency tended to be enhanced by codon optimization, but we were unable to confirm this here. Meanwhile, translational efficiency between PAC and stPAC recombinant genes was defined at the same T3 seed generation by immunoblotting with anti-CRTI antibody (Fig. 4b). The same pattern of signals as in Fig. 3, including two CRTI bands of 55 kDa (CRTI protein alone) and 110 kDa (fusion protein with PSY) and one NSP band 123 Comparison of carotenoid production in a codonoptimized stPAC relative to PAC transgenic rice endosperm Using golden color inspection as the most convenient tool to visualize the carotenoid contents in rice endosperm, color intensity among PAC and stPAC lines was compared at the same T3 seed generation (Fig. 6). Unlike the white endosperm of non-transgenic (NT) rice, a homogeneous yellow color was observed in every transgenic line, with more intense coloring observed among the three stPAC lines relative to the PAC endosperm, indicating the effect of codon optimization on enhanced carotenoid content. The total content and composition of the carotenoids were measured in the same T3 seeds of PAC and stPAC lines by HPLC analysis (Fig. 7; Table S2). The total average amount of carotenoid in the three stPAC lines was 3.5 lg/g (an average of 2.5, 3.4, and 4.6 lg/g), which was Plant Biotechnol Rep Fig. 4 Comparison of transgene expression between PAC and stPAC transformants in T3 seed generation. a qRT-PCR was performed to quantify the relative expression levels in the common 2A-Tp region between PAC and stPAC transcripts. The same amounts of seed RNA were normalized with the OsUbi 5 gene. Error bars represent the Fig. 5 Comparison of transgene expression among stPAC transformants in T3 seed generation. qRT-PCR was performed to quantify the relative expression levels of the stPsy and stCrtI transcripts in three stPAC lines. The same amounts of seed RNA were normalized with the OsUbi 5 gene. Error bars represent the standard deviations among triplicate experimental reactions. NT designates non-transgenic plants used as a negative control Fig. 6 Comparison of seed color phenotypes between PAC and stPAC transformants in T3 seed generation. Photographs showing the color phenotypes of PAC and stPAC transgenic rice seeds along with non-transgenic (NT) rice seeds after dehusking followed by polishing approximately 2.9-time higher than that measured in the PAC line (1.22 lg/g) in Fig. 7a. Interestingly, the b/a ratio of carotenoids in the stPAC lines was 2.7 (an average of 2.3, 2.9, and 2.7), which was far higher than the 1.2 in PAC 4 line and 0.6 in the NT plant (Fig. 7b). This was supported by a big increase in b-carotenoids, including a 4-fold increase in b-carotene (to 2.4 from 0.6 lg/g) and a 2.3-fold increase in zeaxanthin (to 0.16 from 0.07 lg/g) in stPAC lines relative to the PAC 4 line (Fig. 7c) with the altered proportions of b-carotene (68 and 48%) and similarly standard deviations among triplicate experimental reactions. NT designates non-transgenic plants used as a negative control. b Western blot analysis using anti-CRTI antibody to determine the abundance of the CRTI protein were performed as described in Fig. 3 maintaining of zeaxanthin (around 5–6%) between stPAC and PAC 4 lines. As a-carotenoids, a-carotene and lutein also showed a2.1- and 1.5-fold increases in stPAC lines (0.39 and 0.56 lg/g) relative to the PAC 4 line (0.19 and 0.37 lg/g), but no degree of increase was higher than those of the b-carotene (Fig. 7d). The compositional proportions of lutein and a-carotene would rather be decreased in the stPAC (16 and 11%) relative to the PAC 4 (30 and 16%), respectively. Enhanced production of b-carotene component indicates that a codon-optimized stPAC gene had an effect on the enhanced levels of total carotenoids with bbranch preference relative to the PAC gene in rice endosperm. Discussion Codon modification is one tool used to generate the optimum DNA sequence for introduction of heterologous transgenes, which match up with the host conditions, including the codon frequency known as codon bias (Gustafsson et al. 2004; Ikemura 1981; Kanaya et al. 1999; Tuller et al. 2010), GC content (Jackson et al. 2014; Li et al. 2015), and the removal of mRNA destabilizing sequences (Jackson et al. 2014). However, the effectiveness of the gene modified by codon optimization has rarely been reported in comparison with an original unmodified gene, in spite of lots of reports of the development of genetically modified organisms for the production of useful proteins and metabolites using foreign genes (Kim et al. 2016; Li et al. 2016; Liu et al. 2011; Misztal et al. 2004). Among several reports that have adopted the use of synthetic genes in cyanobacteria, Brassica and the rice callus system for the production of isoprene and carotenoids (Bai et al. 2014; Fujisawa et al. 2009; Lindberg et al. 2010), only the DNA sequence of the isoprene synthase gene from kudzu plants was successfully altered for use in 123 Plant Biotechnol Rep Fig. 7 Comparison of carotenoid content and composition between PAC and stPAC transformants in T3 seed generation. HPLC analysis determined the total amount of carotenoid, b/a ratio of carotenoid, and the content of bcarotenoids (b-carotene and zeaxanthin) and a-carotenoids (a-carotene and lutein). Data are expressed as mean (lg/g dry weight) ±SE from three independent experiments using unpolished mature seeds. Numerical results of these figures refer to Table S2 in detail cyanobacteria and resulted in an improvement in protein expression by a factor of about 10 (Lindberg et al. 2010). In this study, we investigated the effect of codon optimization of two genes from Capsicum plants and Pantoea bacteria for use in rice plants, along with analyzing the effect of transgene copy number. The characteristics of the DNA sequence in our final stPAC gene (2,952 bp) were altered by increases in the GC content (58.7 from 50.9%) and CAI (0.85 from 0.77), resulting in 76% DNA similarity based on a PAC gene (2,952 bp). Through the molecular and metabolite analyses of the enough number of transgenic plants (Figs. 1, 2), three chosen stPAC lines showed an average 2.8-fold increase in CRTI translation (Fig. 4b) and more intense yellow color in seeds (Fig. 6) with an average 2.9-fold increase in total carotenoid contents (Fig. 7a; Table S2) relative to those of the PAC 4 line were observed. Interestingly, using a similar approach, the synthetic CrtI gene (Crt-Synth) has been previously examined if b-carotene increased or not (Al-Babili et al. 2006). As a result, this modified gene with 73.8% identity to the original CrtI sequence did not increase the content of carotenoids including b-carotene and CRTI protein abundance (Al-Babili et al. 2006), unlike our results supported by the immunoblot experiments using an anti-CRTI antibody exhibiting enhanced translational efficiency relative to CrtI in PAC (Figs. 3, 4b). This implies that our stCrtI in the stPAC gene was more effectively modified than Crt-Synth. Moreover, the accompanying optimization of codons for the gene encoding PSY as the stPsy might have had a positive influence resulting in increased carotenoid production. 123 To minimize the unintended insertional effects in genetically engineered plants (Schnell et al. 2015), only transgenic lines with a single intact copy of T-DNA were selected and grown under the exact same conditions between PAC and stPAC lines during the coeval T3 generation. 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