Article pubs.acs.org/JAFC
Compositional Equivalency of RNAi-Mediated Virus-Resistant Transgenic Soybean and Its Nontransgenic Counterpart Xiuchun Zhang,†,∥ Pingjuan Zhao,†,∥ Kunxin Wu,† Yuliang Zhang,† Ming Peng,*,†,‡ and Zhixin Liu*,† †
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Haikou, China ‡ Environmental Safety Supervision and Inspection Centre for Genetically Modified Plants and Microorganisms Used in Plants, Ministry of Agriculture, Haikou, China ABSTRACT: RNA silencing or RNA interference (RNAi), which is triggered by double-stranded RNA (dsRNA), is an evolutionarily conserved process that is active in a wide variety of eukaryotic organisms. Engineering plants with hairpin construct in which the viral gene is arranged in inverted repeats (IR) renders plants resistant to plant virus infection. However, there is no report on whether biologically important changes occurred by the insertion of IR, which confer transgenic plants virus resistance. In the present study, the compositions of virus-resistant transgenic soybean seeds developed by insertion of three short IRs, each containing the specific, highly conserved sequences derived from one virus, were compared with those of nontransgenic counterparts by applying the principle of substantial equivalence to determine whether significant undesirable biological changes occurred by IR insertion. The results revealed that the nutrient components as well as antinutrient contents of these virusresistant soybean lines are substantially equivalent to those of the nontransgenic counterparts, and the majority of the measured amounts of nutritional components and antinutrient contents are well within the range of values reported for other commercial soybean lines. The results imply that no biologically important changes occurred by the insertion of IRs in the RNAi-mediated virus-resistant transgenic soybeans. The results can serve as baseline information for developing RNAi-mediated transgenic soybean cultivars or other crops with broader spectrum virus resistance. KEYWORDS: inverted repeats, virus-resistant transgenic soybean, composition, substantial equivalence
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INTRODUCTION Research during the past decade has firmly established that land plants use an RNA-targeting defense mechanism termed RNA silencing (or RNA interference, RNAi) to combat virus infections. The trigger of RNA silencing is a double-stranded RNA (dsRNA), which is processed into small interfering RNAs (siRNAs), which direct Argonaute protein silencing corresponding genes or genetic elements in a sequence-specific manner.1,2 On the basis of RNAi machinery, engineering plants with hairpin construct in which the transgenes are arranged in inverted repeats (IR) offers a promising alternative for controlling most major virus disease problems in crops.3 Considering the history of traditional breeding on safety, the likelihood that safety concern might arise due to unintended compositional changes resulting from transgenes is negligible. However, compositional studies continue to be required, and regulatory requirements for composition studies are becoming increasingly complex in some regions.4 Soybean (Glycine max L. Merrill) is an important staple source of vegetable protein in food and animal feed as well as for industry because the abundance and nutritional quality of seed proteins in soybean exceed those in other grain crops. To our best knowledge, there is no report on whether biologically important changes occurred by the insertion of IR that conferred transgenic plants virus resistance. The present study was designed to evaluate the composition of the RNAi-mediated virus-resistant transgenic soybeans engineered with hairpin construct and their nontransgenic counterpart through a series of chemical analyses to determine whether significant undesirable biologically © 2014 American Chemical Society
changes occurred by the IR insertion in transgenic soybeans, using the concept of “substantial equivalence”, which was developed by the Organisation for Economic Co-operation and Development (OECD) in 1993 and was elaborated further by FAO/WHO.5,6 The concept suggests that if no meaningful difference from the conventional counterpart is found, then the transgenic crop is as safe and nutritious as its traditional counterpart, which is generally accepted as safe on the basis of human food use history. Substantial equivalent analysis has been studied for different events of transgenic crops, such as corn,7,8 soybean,9−15 rice,16 and papaya.17
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MATERIALS AND METHODS
Soybean Used for Analysis. The transgenic soybeans and respective nontransgenic counterparts were cultivated in cultivation pots, which were 30 cm in diameter and 23 cm in height, in the greenhouse of the Chinese Academy of Tropical Agriculture in Hainan, China. Each cultivar (one transgenic soybean line or its nontransgenic line) was composed of 10 cultivation pots of soybeans. The arrangement of soybean cultivation pots was randomly assigned. The transgenic soybean lines were T4 generation of self-crossing progenies from T0 generation of soybean transformants, which were developed through the Agrobacterium-mediated transformation with transgene construct dsABS.18 The construct contains three hairpins, which derived from the highly conserved regions of replicase genes of Received: Revised: Accepted: Published: 4475
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Table 1. Proximate Compositiona of Virus-Resistant Transgenic Soybean and Nontransgenic Counterpart Seeds (Mean ± SD; n = 3)
a
component
Throne
729-3
729-4
729-5
lit. rangeb
moisture ash crude fat crude protein carbohydrate
7.06 ± 0.08 4.73 ± 0.06 13.81 ± 0.11 37.28 ± 0.15 37.12 ± 0.16
7.11 ± 0.05 4.69 ± 0.09 13.62 ± 0.19 37.43 ± 0.12 37.15 ± 0.11
7.08 ± 0.06 4.66 ± 0.04 13.87 ± 0.13 37.37 ± 0.09 37.02 ± 0.14
7.05 ± 0.03 4.72 ± 0.07 13.66 ± 0.17 37.33 ± 0.18 37.24 ± 0.19
7−11 4.61−5.37 13.2−22.5 36.9−46.4 22.0−46.1
Grams per 100 g of dry weight (moisture based on fresh weight). bFrom ref 29.
Table 2. Amino Acid Compositiona of Virus-Resistant Transgenic Soybean and Nontransgenic Counterpart Seeds (Mean ± SD; n = 3) component alanine arginine asparagine cysteine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine a
Throne 1.70 2.55 4.25 0.58 6.99 1.66 0.85 1.77 2.88 2.45 0.56 1.88 1.94 1.81 1.59 0.54 1.18 1.78
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.10 0.01 0.13 0.02 0.01 0.02 0.05 0.03 0.02 0.01 0.01 0.02 0.01 0.02 0.01 0.02
729-3 1.78 2.57 4.35 0.57 7.13 1.65 0.82 1.79 2.97 2.49 0.57 1.91 2.02 1.82 1.63 0.56 1.16 1.71
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
729-4
0.04 0.03 0.09 0.02 0.15 0.01 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.02 0.01 0.03 0.01
1.72 2.64 4.38 0.59 7.10 1.69 0.81 1.82 2.93 2.54 0.55 1.90 1.99 1.81 1.60 0.56 1.15 1.73
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.02 0.11 0.02 0.14 0.03 0.03 0.02 0.02 0.02 0.03 0.02 0.01 0.02 0.01 0.02 0.03 0.02
729-5
lit. rangeb
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.21−1.72 1.91−3.13 3.02−4.66 0.34−0.72 4.78−7.40 1.19−1.74 0.75−1.08 1.21−1.90 2.15−3.15 1.73−2.66 0.37−0.61 1.36−2.05 1.31−2.12 0.98−2.28 1.01−1.51 0.28−0.47 0.87−1.43 1.24−2.05
1.74 2.59 4.39 0.58 6.97 1.71 0.84 1.80 2.85 2.48 0.56 1.86 1.96 1.85 1.58 0.55 1.20 1.75
0.03 0.02 0.10 0.02 0.12 0.02 0.02 0.03 0.06 0.02 0.01 0.03 0.03 0.02 0.02 0.02 0.01 0.02
Grams per 100 g of dry weight. bFrom ref 29. from proteins by heating at approximately 110 °C in 4.2 M sodium hydroxide for 20 h. After acid hydrolysis, the sample was separated on an anion exchange column and detected with a ninhydin reaction as described in the literature.23 Fatty Acid Profile. Up to 20 g of ground powder of transgenic or nontransgenic soybean seeds was analyzed through gas chromatography according to the procedures from the literature.24 Antinutrients. Lectins were determined by measuring the agglutinating properties of soybean sample extract on rabbit red blood cells according to modified procedures from the literature.25,26 Trypsin inhibitor activity was determined by using a modified method described previously in the literature.27 Isoflavones Profile. The profiles of isoflavones of soybean seeds were determined through liquid chromatography according to procedures from the literature.28 Statistical Analysis. After harvest, the seeds of 10 cultivation pots of each cultivar were put together. Half of each cultivar’s harvested dried seeds, which were selected randomly, were milled into powder using a high-speed tissue masher for nutrient compositional analysis. All statistical data analyses were performed employing the SPSS 19.0 software package. All analytical determinations were conducted in triplicate, and results are expressed as the mean ± standard deviation. Differences between samples were tested using one-way analysis of variance (ANOVA). P ≤ 0.05 was considered to be statistically significant.
Alfalfa mosaic virus (AMV, a member of the family Bromoviridae), Bean pod mottle virus (BPMV, a member of the family Secoviridae), and Soybean mosaic virus (SMV, a member of the family Potyviridae), respectively, and the bar gene isolated from Streptomyces hygroscopicus as a selectable marker. Compositional Analyses. Proximate. Moisture content was determined gravimetrically by drying the samples in a vacuum oven at approximately 100 °C.19 For ash determination, the samples were placed in an electric furnace at 550 °C and ignited in air. The residual ash was quantified gravimetrically.20 The Kjeldahl method was employed to determine total nitrogen and protein of the sample. The percent nitrogen was calculated and converted to equivalent protein using a factor of 6.25.21 The crude fat content of the soybean seed samples was determined using the Soxhlet extraction method.22 The carbohydrates content was calculated by difference using the fresh weight-derived data according to the following equation:
% carbohydrates = 100 − (% protein + % fat + % ash + % moisture) Amino Acid Profile. Protein hydrolysis was performed before ninhydrin derivatization to analyze total amino acid composition, with the exception of cysteine, methionine, and tryptophan. The samples were hydrolyzed with 1 mL of 6 M hydrochloric acid at 110 °C for 22 h under a nitrogen atmosphere. The hydrolysate was dried under reduced pressure, dissolved in 0.02 M HCl, and subjected to analysis using a UV detector and a Hitachi L-8800 automatic amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan). The cystine and cysteine in the samples were oxidized to cysteic acid, and methionine was oxidized to methionine sulfone through dithiodipropionic acid treatment for 16 h at approximately 0 °C. Tryptophan is hydrolyzed
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RESULTS AND DISCUSSION Proximate Compositions. Table 1 shows the proximate analyses, including moisture, ash content, crude protein, crude fat, and carbohydrates, of the three virus-resistant transgenic 4476
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Table 3. Fatty Acid Compositiona of Virus-Resistant Transgenic Soybean and Nontransgenic Counterpart Seeds (Mean ± SD; n = 3) fatty acid 14:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:1 22:0 24:0 a
myristic palmitic palmitoleic heptadecanoic heptadecenoic stearic oleic linoleic linolenic arachidic eicosenoic behenic lignoceric
Throne 0.079 10.8 0.082 0.116 0.065 4.2 20.2 55.1 8.4 0.33 0.17 0.35 0.12
± ± ± ± ± ± ± ± ± ± ± ± ±
0.003 0.13 0.002 0.003 0.002 0.22 1.32 2.43 0.35 0.01 0.01 0.02 0.01
729-3 0.080 10.6 0.084 0.119 0.062 4.2 19.9 55.8 8.6 0.33 0.17 0.34 0.11
± ± ± ± ± ± ± ± ± ± ± ± ±
729-4
0.003 0.13 0.003 0.002 0.002 0.22 1.38 2.49 0.35 0.02 0.01 0.02 0.01
0.079 10.7 0.083 0.119 0.065 4.2 19.8 55.6 8.5 0.33 0.17 0.35 0.12
± ± ± ± ± ± ± ± ± ± ± ± ±
0.003 0.15 0.003 0.002 0.003 0.22 1.32 2.43 0.37 0.02 0.01 0.02 0.01
729-5
lit. rangeb
± ± ± ± ± ± ± ± ± ± ± ± ±
0.071−0.238 9.55−15.77 0.086−0.194 0.085−0.146 0.073−0.087 2.70−5.88 14.3−32.2 42.3−58.8 3.00−12.52 0.16−0.48 0.14−0.35 0.163−0.482
0.079 10.6 0.082 0.118 0.064 4.1 20.1 55.1 8.5 0.32 0.17 0.35 0.12
0.003 0.13 0.003 0.001 0.002 0.22 1.32 2.53 0.35 0.02 0.01 0.02 0.02
Percent total fatty acid. bFrom ref 29.
Table 4. Trypsin Inhibitor and Lectin Composition of Virus-Resistant Transgenic Soybean and Nontransgenic Counterpart Seeds (Mean ± SD; n = 3) component
Throne
729-3
729-4
729-5
lit. rangec
lectin (HU/mg)a trypsin inhibitor (TIU/mg)b
6.06 ± 0.08 33.15 ± 0.12
6.05 ± 0.10 32.98 ± 0.16
6.10 ± 0.11 34.01 ± 0.14
6.09 ± 0.15 33.57 ± 0.15
0.09−8.46 18−108
a
Fresh weight basis. HU, hemagglutinating units. HU/mg denotes lectin units per milligram of fresh weight of soybean seeds. bTIU, trypsin inhibitor units. cFrom ref 29.
Table 5. Contentsa of Isoflavones in Virus-Resistant Transgenic Soybean and Nontransgenic Counterpart Seeds (Mean ± SD; n = 3)
a
isoflavone
Throne
729-3
729-4
729-5
lit. rangeb
daidzin glycitin genistin daidzein glycitein genistein
81.6 ± 0.6 13.3 ± 0.4 100 ± 1.2 8.9 ± 0.2 4.0 ± 0.1 8.8 ± 0.2
82.2 ± 0.5 12.9 ± 0.6 102 ± 1.1 8.8 ± 0.1 4.1 ± 0.0 8.9 ± 0.1
81.8 ± 0.5 13.4 ± 0.6 99 ± 1.3 9.1 ± 0.3 4.1 ± 0.1 8.8 ± 0.0
82.1 ± 0.7 13.2 ± 0.5 98 ± 1.0 8.9 ± 0.2 3.9 ± 0.2 8.9 ± 0.1
35.8−360 10.5−46.7 74.8−442.3 2.2−47.7 1.1−6.2 1.5−18.3
Micrograms per gram dry weight. bFrom ref 30.
amino acid contents of the transgenic soybean cultivars were substantially equivalent to those of nontransgenic counterparts. Fatty Acids. Individual fatty acids of soybean samples were estimated by GC-MS analysis. All fatty acids were expressed as percentage with respect to the total fatty acids (Table 3). Among them, consistent with previous results,14−20 palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:3) were the most abundant fatty acids of transgenic virus-resistant soybeans and their nontransgenic counterpart. The percentage values of the majority of the measured fatty acids fell within the ranges of the ILSI database,29 except palmitoleic and heptadecenoic; the contents of these two fatty acids were lower than the minimum values. However, with a confidence level of 95% no statistical differences were found in fatty acid composition. The contents of the fatty acids in harvested seeds of the virus-resistant soybeans 729-3, 729-4, and 729-5 were comparable to those observed in the seeds of conventional control. Antinutrients. The results for two important antinutritional factors (lectin and trypsin inhibitors) are presented in Table 4. Both of them were generally low and did not exceed the range reported previously,29 which might not affect the nutritional potential of soybean seeds. The contents of lectin and trypsin
soybean lines and their nontransgenic counterpart seeds. In terms of these compositions, with a confidence level of 95%, no statistically significant differences were observed not only between the transgenic soybean seeds and the control but also among the different transgenic soybean lines. Individual values were within the tolerance interval determined for commercial varieties and within published literature ranges.29 These results implied that no biologically important changes occurred for the proximate compositions of the transgenic soybean lines 729-3, 729-4, and 729-5 compared with their nontransgenic counterpart. Amino Acid Contents. Table 2 presents the content of 18 amino acids of transgenic virus-resistant soybean and nontransgenic seeds. The content of individual amino acids in soybean seeds was expressed as percentage with respect to the total protein. The contents of the majority of the measured amino acids fell within the ranges of the ILSI database,29 except threonine and tryptophan; the contents of these were higher than the maximum values. However, no significant differences in the levels of any of the 18 amino acids measured were found between the virus-resistant soybean seeds and their nontransgenic counterparts. These results demonstrated that the 4477
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Technology Program of Hainan Province (ZDZX2013010-3 and ZDZX2013023-1).
inhibitor in the harvested seeds of virus-resistant transgenic soybeans were comparable with those observed in the harvested seeds of the nontransgenic control with a confidence level of 95%. These results exhibited that the antinutrient composition of the transgenic lines was substantially equivalent to that of the nontransgenic counterpart. Isoflavones Profile. The contents of isoflavones including daidzin, glycitin, genistin, daidzein, glycitein, and genistein in the harvested seeds of virus-resistant transgenic soybeans were comparable to those observed in the harvested seeds of the conventional control (Table 5). The level of these isoflavones fell within the ranges of other commercially available soybeans reported in previous literature.30 No significant statistical difference was observed in any of the isoflavones levels between the transgenic and nontransgenic soybean seeds. In conclusion, the results of compositional analyses show no meaningful differences had been found in the transgenic soybean lines with glyphosate tolerance and virus resistance from the conventional counterpart with a confidence level of 95%. On the basis of the principle of substantial equivalence,5,6 the transgenic soybean lines with glyphosate tolerance and virus resistance are substantially equivalent to those nontransgenic counterparts. The results reported herein, together with previous findings that the composition of transgenic glyphosate-tolerant soybeans with the bar gene is equivalent with that of conventional soybeans,9−15 support the conclusion that no significant undesirable biological changes occurred by the insertion of IR conferring transgenic soybean with robust resistance to mixed infection of AMV, BPMV, and SMV. RNA silencing or RNA interference (RNAi), a form of gene suppression that occurs at transcriptional and post-transcriptional levels, is an evolutionarily conserved process that is active in a wide variety of eukaryotic organisms.1,2 One of the advantages of RNAi-mediated virus resistance is the relatively small size of the transgene required for silencing, enabling more than one short IR containing different viruses’ conserved sequences to be stacked to a single construct and enabling a broader spectrum of resistance to be developed with a single transgene.3 RNAi has been an important tool to render plants resistant to plant virus infection,31 although there are three main factors that can determine the practical usefulness of antiviral strategies: efficiency, durability, and safety.32 The results showed that the transgenic soybeans with IR insertion are substantially equivalent to nontransgenic counterparts. The concept of “substantial equivalence” suggests that the RNAimediated virus-resistant transgenic soybeans are as safe and nutritious as their traditional counterpart. The results can serve as baseline information for developing RNAi-mediated transgenic soybean cultivars or other crops with broader spectrum virus resistance.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We greatly appreciate the generosity of Feng Qu at The Ohio State University for providing the experimental material for this study.
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AUTHOR INFORMATION
Corresponding Authors
*(M.P.) Phone: +86-898-66890770. Fax: +86-898-66890978. E-mail:
[email protected]. *(Z.L.) Phone: +86-898-66890770. Fax: +86-898-66890978. Email:
[email protected]. Author Contributions ∥
REFERENCES
(1) Ding, S. RNA-based antiviral immunity. Nat. Rev. Immunol. 2010, 10 (9), 632−644. (2) Qu, F. Plant viruses versus RNAi: simple pathogens reveal complex insights on plant antimicrobial defense. Wiley Interdiscip. Rev. RNA 2010, 1, 22−23. (3) Mansoor, S.; Amin, I.; Hussain, M.; Zafar, Y.; Briddon, R. W. Engineering novel traits in plants through RNA interference. Trends Plant Sci. 2006, 11 (11), 559−565. (4) EFSA Panel on Genetically Modified Organisms (GMO). Statistical considerations for the safety evaluation of GMOs. EFSA J. 2010, 8 (1), 1250. (5) OECD (Organisation for Economic Cooperation and Development). Concepts and Principles; Paris, France, 1993. (6) FAO/WHO. Evaluation of Allergenicity of Genetically Modified Foods; report of a joint FAO/WHO expert consultation on allergenicity of foods derived from biotechnology; Rome, Italy, 2001. (7) Tang, M.; He, X.; Luo, Y.; Ma, L.; Tang, X.; Huang, K. Nutritional assessment of transgenic lysine-rich maize compared with conventional quality protein maize. J. Sci. Food Agric. 2013, 93 (5), 1049−1054. (8) George, C.; Ridley, W. P.; Obert, J. C.; Nemeth, M. A.; Breeze, M. L.; Astwood, J. D. Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn (Zea mays L.). J. Agric. Food Chem. 2004, 52 (13), 4149−4158. (9) Qin, F.; Kang, L.; Guo, L.; Lin, J.; Song, J.; Zhao, Y. Composition of transgenic soybean seeds with higher γ-linolenic acid content is equivalent to that of conventional control. J. Agric. Food Chem. 2012, 60 (9), 2200−2204. (10) Berman, K. H.; Harrigan, G. G.; Riordan, S. G.; Nemeth, M. A.; Hanson, C.; Smith, M.; Sorbet, R.; Zhu, E.; Ridley, W. P. Compositions of forage and seed from second-generation glyphosate-tolerant soybean MON 89788 and insect-protected soybean MON 87701 from Brazil are equivalent to those of conventional soybean (Glycine max). J. Agric. Food Chem. 2010, 58 (10), 6270− 6276. (11) Lepping, M. D.; Herman, R. A.; Potts, B. L. Compositional equivalence of DAS-444Ø6-6 (AAD-12 + 2mEPSPS + PAT) herbicide-tolerant soybean and nontransgenic soybean. J. Agric. Food Chem. 2013, 61 (46), 11180−11190. (12) Berman, K. H.; Harrigan, G. G.; Nemeth, M. A.; Oliveira, W. S.; Berger, G. U.; Tagliaferro, F. S. Compositional equivalence of insectprotected glyphosate-tolerant soybean MON 87701 × MON 89788 to conventional soybean extends across different world regions and multiple growing seasons. J. Agric. Food Chem. 2011, 59 (21), 11643− 11651. (13) Herman, R. A.; Phillips, A. M.; Lepping, M. D.; Sabbatini, J. Compositional safety of DAS-68416-4 (AAD-12) herbicide-tolerant soybean. J. Nutr. Food Sci. 2011, DOI: 10.4172/2155-9600.1000103. (14) Taylor, N. B.; Fuchs, R. L.; MacDonald, J.; Shariff, A. R.; Padgette, S. R. Compositional analysis of glyphosate-tolerant soybeans treated with glyphosate. J. Agric. Food Chem. 1999, 47 (10), 4469− 4473. (15) Harrigan, G. G.; Ridley, W. P.; Riordan, S. G.; Nemeth, M. A.; Sorbet, R.; Trujillo, W. A.; Breeze, M. L.; Schneider, R. W. Chemical composition of glyphosate-tolerant soybean 40-3-2 grown in Europe
X.Z. and P.Z. contributed equally to this work.
Funding
This work was financially supported by the Natural Science Foundation of China (31201264) and the Major Science and 4478
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remains equivalent with that of conventional soybean (Glycine max L.). J. Agric. Food Chem. 2007, 55 (15), 6160−6168. (16) Gayen, D.; Sarkar, S. N.; Datta, S. K.; Datta, K. Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart. Food Chem. 2013, 138 (2−3), 835−840. (17) Roberts, M.; Minott, D. A.; Pinnock, S.; Tennant, P. F.; Jackson, J. C. Physicochemical and biochemical characterization of transgenic papaya modified for protection against papaya ringspot virus. J. Sci. Food Agric. 2014, 94, 1034−1038. (18) Zhang, X.; Sato, S.; Ye, X.; Dorrance, A. E.; Morris, T. J.; Clemente, T. E.; Qu, F. Robust RNAi-Based resistance to mixed Infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. Phytopathology 2011, 101 (11), 1264−1269. (19) GB 5009.3-2010. Determination of moisture in foods. Standardization Administration of the People’s Republic of China, Beijing, China, 2010. (20) GB 5009.4-2010. Determination of ash in foods. Standardization Administration of the People’s Republic of China, Beijing, China, 2010. (21) GB 5009.5-2010. Determination of protein in foods. Standardization Administration of the People’s Republic of China, Beijing, China, 2010. (22) GB 5009.6-2003. Determination of fat in foods. Standardization Administration of the People’s Republic of China, Beijing, China, 2003. (23) GB/T 5009.124-2003. Determination of amino acids in foods. Standardization Administration of the People’s Republic of China, Beijing, China, 2003. (24) GB/T 17377-2008. Animal and vegetable fats and oils − analysis by gas chromatography of methyl esters of fatty acids. Standardization Administration of the People’s Republic of China, Beijing, China, 2008. (25) Klurfeld, D. M.; Kritchevski, D. Isolation and quantitation of lectins from vegetable oils. Lipids 1987, 22, 667−668. (26) Martin, S.; Jost, W.; Hans-Curt, F. Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J. Microbiol. Methods 2002, 50, 237−248. (27) Hamerstrand, G. E.; Black, L. T.; Glover, J. D. Trypsin inhibitors in soy products: Modification of the standard analytical procedure. Cereal Chem. 1981, 58, 42−45. (28) GB/T 23788-2009. Determination of soybean isoflavone in health-care food − High-performance liquid chromatography. Standardization Administration of the People’s Republic of China, Beijing, China, 2009. (29) ILSI International Life Sciences Institute (ILSI) Crop Composition Database, version 4.0; http://www.cropcomposition. org (search criteria: soybean seed, all locations, all years) (accessed Feb 10, 2014). (30) Kim, J. K.; Kim, E. H.; Park, I.; Yu, B. R.; Lim, J. D.; Lee, Y. S.; Lee, J. H.; Kim, S. H.; Chung, I. M. Isoflavones profiling of soybean [Glycine max (L.) Merrill] germplasms and their correlations with metabolic pathways. Food Chem. 2014, 153, 258−264. (31) Collinge, D. B.; Jørgensen, H. J.; Lund, O. S.; Lyngkjaer, M. F. Engineering pathogen resistance in crop plants: current trends and future prospects. Annu. Rev. Phytopathol. 2010, 48, 269−291. (32) Simón-Mateo, C.; García, J. A. Antiviral strategies in plants based on RNA silencing. Biochim. Biophys. Acta 2011, 1809 (11−12), 722−731.
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