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, 11 (10), e0163352
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Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats Through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota

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Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats Through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota

Kai Zhao et al. PLoS One.

Abstract

In this study, assessment of the safety of transgenic rice T1C-1 expressing Cry1C was carried out by: (1) studying horizontal gene transfer (HGT) in Sprague Dawley rats fed transgenic rice for 90 d; (2) examining the effect of Cry1C protein in vitro on digestibility and allergenicity; and (3) studying the changes of intestinal microbiota in rats fed with transgenic rice T1C-1 in acute and subchronic toxicity tests. Sprague Dawley rats were fed a diet containing either 60% GM Bacillus thuringiensis (Bt) rice T1C-1 expressing Cry1C protein, the parental rice Minghui 63, or a basic diet for 90 d. The GM Bt rice T1C-1 showed no evidence of HGT between rats and transgenic rice. Sequence searching of the Cry1C protein showed no homology with known allergens or toxins. Cry1C protein was rapidly degraded in vitro with simulated gastric and intestinal fluids. The expressed Cry1C protein did not induce high levels of specific IgG and IgE antibodies in rats. The intestinal microbiota of rats fed T1C-1 was also analyzed in acute and subchronic toxicity tests by DGGE. Cluster analysis of DGGE profiles revealed significant individual differences in the rats' intestinal microbiota.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Safety assessment of HGT.
PCR analysis of cry1C gene from masseter muscle (a), duodenum (b), ileum (c). M, DL2000 marker; 1–3, genomic DNA of T1C-1, Minghui 63 and control group, respectively; 4–6, prl of the genomic DNA of T1C-1, Minghui 63 and control group, respectively; 7, 35S promoter positive control; 8–10, 35S promoter of T1C-1, Minghui 63 and control group, respectively; 11, NOS terminator positive; 12–14, NOS terminator of T1C–1, Minghui 63 and control group, respectively; 15, exogenous gene control; 16–18, exogenous gene cry1C of T1C-1, Minghui 63 and control group, respectively. PCR analysis of cry1C gene from microbes in anaerobic cultures (d), Salmonella (e), Lactobacilli (f), Streptococcus (g), E. coli (h). M, DL2000 marker; 1–3, genomic DNA of T1C-1, Minghui 63 and control group, respectively; 4–6, bacterial 16S DNA of T1C-1, Minghui 63 and control group, respectively; 7, 35S promoter positive control; 8–10, 35S promoter of T1C-1, Minghui 63 and control group, respectively; 11, NOS terminator positive; 12–14, NOS terminator of T1C–1, Minghui 63 and control group, respectively; 15, exogenous gene control; 16–18, exogenous gene cry1C of T1C-1, Minghui 63 and control group, respectively.
Fig 2
Fig 2. SDS-PAGE, western blot analysis of Cry1C protein and the protein degradation test in SGF and SIF.
(a) M, molecular mass protein standards; 1–3, lysis supernatant of Transetta (DE3) cells, cells transformed with pET30a (+) and pET30a–Cry1C, respectively; 4, 5, Cry1C fusion protein. (b) Western blot analysis of Cry1C protein expressed in E. coli (lane 1) and the rice-derived Cry1C protein (lane 2). Gel staining (c,e) and western blot analysis (d,f) of fusion protein digested with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), respectively. M, molecular mass protein standards; 1–8, 0 s, 15 s, 30 s, 60 s, 2 min, 10 min, 30 min and 60 min after digestion, respectively.
Fig 3
Fig 3. DGGE profiles and analysis by UPGAMA from rat fecal bacteria in the control and test group, respectively.
(a) 1–4, 5–8, 9–12, samples from R1, R2 and R3 (the three rats in the control group) at 0, 1, 7 and 14 d after gavage respectively. (b) 1–4, 5–8, 9–12, samples from T1, T2 and T3 (the three rats in the test group) at 0, 1, 7 and 14 d, respectively. (c) 1–4, 5–8, 9–12, Samples from T4, T5, T6 (the other three rats in the test group) at 0, 1, 7 and 14 d, respectively. (d–f) analysis of rat fecal samples by UPGAMA after gavage.
Fig 4
Fig 4. DGGE profiles and cluster analysis of the profiles from rat fecal bacteria.
(a,b) DGGE profiles in test group fed daily with Bt rice T1C-1 expressing Cry1C. (c,d) Cluster analysis of DGGE profiles of a, b. (e,f) DGGE profiles in control group fed with rice Minghui 63. (g,h) Cluster analysis of DGGE profiles of g, h. (i,j) DGGE profiles in control group fed with untreated food. (k,l) Cluster analysis of DGGE profiles of i, j. a, 1–6, samples from test group A1; 7–12, samples from A2; 13–18, samples from A3 at 0, 30, 60, 80, 85 and 90 d after feeding with transgenic rice T1C-1 expressing Cry1C. b, 1–6, samples from A4; 7–11, samples from A5; 12–18, samples from A6 at 0, 30, 60, 80, 85 and 90 d after feeding with transgenic rice T1C-1 expressing Cry1C. e: 1–6: samples from control group B1; 7–12: samples from B2; 13–18: samples from B3 at 0, 30, 60, 80, 85 and 90 d after feeding with rice Minghui 63; f: 1–6: samples from B4; 7–12: samples from B5; 13–18: samples from B6 at 0, 30, 60, 80, 85 and 90 d after feeding with rice Minghui 63. i: 1–6: samples from control group fed untreated food daily C1; 7–12: samples from C2; 13–18: samples from C3 at 0, 30, 60, 80, 85 and 90 d after feeding with untreated food daily; j: 1–6: samples from C4; 7–12: samples from C5; 13–18: samples from C6 at 0, 30, 60, 80, 85 and 90 d after feeding with untreated food daily.
Fig 5
Fig 5. Shannon index (H) and Simpson’s index (D) of each band from DGGE bands and phylogenetic tree analysis of the 16S rDNA gene sequences of the obtained clones.
(a,b,c) A1–A6, B1–B6 and C1–C6 have the same meanings as for Fig 4, ■The value of H, ▲ the value of D. (d) GenBank accession numbers are given in parentheses. The scale bar indicates the 0.05 evolutionary distance unit. Bootstrap values (percent) are shown at branch nodes.

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Grant support

This work was supported by Shanghai Agricultural Science Committee foundation of China, grant no.6-4 (2009),http://e-nw.shac.gov.cn/index.html. Shanghai Agricultural Science Key Research Foundation grant no.1-8 (2011), http://e-nw.shac.gov.cn/index.html. The Public Research Platform Foundation of Shanghai Science and Technology Committee, grant no. 13DZ2290700, http://www.stcsm.gov.cn/. The Science and Technology Development Programme of Shanghai Acedemy of Agricultural Sciences,2013(14), http://www.saas.sh.cn/. The Science and Technology Development Programme of Shanghai Acedemy of Agricultural Sciences,2012(8), http://www.saas.sh.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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