Effects of genetically modified T2A-1 rice on the GI health of rats after 90-day supplement. Huang 1,2,* and Yunbo Luo 1,2

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1 Effects of genetically modified T2A-1 rice on the GI health of rats after 90-day supplement Yanfang Yuan 1, Wentao Xu 1,2, *, Xiaoyun He 2, Haiyan Liu 2, Sishuo Cao 1, Xiaozhe Qi 1, Kunlun Huang 1,2,* and Yunbo Luo 1,2 1 Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing , China 2 The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing , China * Corresponding author

2 Figure legends Fig S1. Growth curves and daily feed intakes. (A) Growth curves for both male and female rats. Curves based on weekly measurements of body weights are shown with means based on six rats/sex/group; (B-1) Mean relative food intake of male rats (n=6); (B-2) Mean relative food intake of female rats (n=6). : Male rats; : Female rats. FigS2. Effects of diets on intestinal tight-junction proteins Tight-junction protein changes in ileum was measured with Zo-1 in (A) AIN93G group, (B) genetically modified (GM) group, (C) nongenetically modified (NM) group, and Occludin in (E) AIN93G group, (F) GM group, (G) NM group, and (D) experimental control without the addition of secondary antibody. Table S1. Organ weights. Table S2. Results of hematology analysis. Table S3. Bacteria composition of content from different parts of the gastrointestinal (GI) tract. Table S4. ph and water content in fecal and cecum contents. Table S5. Effect of cry2a rice on short-chain fatty acid (SCFA) concentration (umol/g) in cecum and feces of rats. Table S6. Primers used in this study.

3 Supplementary Tables Table S1 Absolute weights Male Female CK NM GM CK NM GM Body weight ± ± ± ± ± ±24.79 Adrenals 0.092± ± ± ± ± ±0.023 Brains 2.25± ± ± ± ±0.06 * 1.89±0.09 * Heart 1.93± ± ± ± ± ±0.11 Kidneys 3.67± ± ± ± ± ±0.07 Liver 13.12± ± ± ± ± ±0.79 Lung 1.66± ±0.12 * 1.78± ± ± ±0.59 Spleen 0.74± ± ±0.08 * 0.16± ± ±0.03 Testis 3.43± ± ± ± ± ±0.08 Thymus 0.38± ± ± ± ± ±0.05 Relative weights Adrenals 4.58± ± ± ± ± ±1.16 Brains 0.44± ± ± ± ± ±0.07 Heart 0.37± ± ± ± ± ±0.04 Kidneys 0.71± ± ± ± ± ±0.07 Liver 2.54± ± ± ± ± ±0.44 Lung 0.32± ±0.03 * 0.34± ± ± ±0.18 Spleen 0.14± ± ±0.02 * 0.05± ± ±0.01 Testis 0.66± ± ± ± ± ±0.04 Thymus 0.08± ± ± ± ± ±0.02 Results are showed in mean values ± SD. *:Statistically significant differences from the CK group (P < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group.

4 Table S2 Routine analysis of blood Male Female CK NM GM CK NM GM WBC (*10 9 /L) 8± ± ± ± ± ±2.38 RBC (*10 12 /L) 7± ± ± ± ± ±0.44 PLT (*10 9 /L) ± ± ± ± ± ±25.61 HGB (g/l) 203± ± ± ± ± ±37.69 HCT (%) 52.27± ± ± ± ± ±3.77 MCV (fl) 74.72± ± ± ± ± ±1.02 MCH (pg) 29.02± ± ± ± ± ±0.98 MCHC (g/l) ± ± ± ± ± ±12.48 Differential count Lymphocytes (%) 75.17± ± ± ± ± ±12.88 Neutrophils (%) 23± ± ± ± ± ±12.73 Eosinophils (%) 1.17± ± ± ±1.63 2± ±1.75 Basophils (%) 0±0 0±0 0±0 0±0 0±0 0±0 Monocytes (%) 0.67±0.82 0±0 0.5± ±0.52 0±0 0.5±0.84 Blood biochemistry TP (mmol/l) 7.03± ± ± ± ± ±0.92 ALT (U/L) 80.78± ±12.6 * 58.77±7.45 * 77.92± ± ±19.74 AST (U/L) ± ± ± ± ± ±45.84 ALP (U/L) ± ± ± ± ± ±5.40 ALB (g/l) 39.71± ± ±1.31 * 53.82± ± ±4.97 BUN (mmol/l) 7.06± ± ± ± ± ±2.09 CREA (μmol/ml) 29.41± ± ± ± ± ±2.91 T-CHO (mmol/l) 2.32± ± ± ± ± ±0.40 * TG (mmol/l) 1.23± ± ± ± ± ±0.41 GLU (mmol/l) 8.18± ± ± ± ± ±1.19 P (mmol/l) 2.87± ± ± ± ± ±0.19 Ca (mmol/l) 2.83± ± ± ± ± ±0.10 Results are showed in mean values ± SD. *:Statistically significant differences from the CK group (P < 0.05) :Statistically significant difference from the parental line (P < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group; WBC:white blood cells; RBC: red blood cells, PLT: platelets, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume; MCHC: Mean Corpuscular Hemoglobin Concentration; TP: total protein; ALT: alanine transaminase; AST: aspartate transaminase; ALP: alkaline phosphatase; ALB: albumin; BUN: blood urea nitrogen; CREA: creatinine; T-CHO: total cholesterol; TG: triglyceride; GLU: glucose; Ca: calcium, P: inorganic phosphorus.

5 Table S3 Lactobacilli Bifidobacteria Bacteroides Coliforms Enterococci Clostridium Total bacteria B/E bacteria-day 30 CK 8.38± ± ± ± ± ± ± NM 8.87±0.28 * 8.86± ± ± ±0.57 * 6.16± ± GM 8.82±0.34 * 8.71± ± ± ±0.31 * 6.13± ± CK 8.45± ± ± ± ± ± ± NM 8.90±0.37 * 8.60± ± ± ±0.48 * 6.24± ± GM 8.93±0.41 * 8.76± ± ± ±0.55 * 6.10± ± bacteria-day 60 CK 8.61± ± ± ± ± ± ± NM 8.82± ± ± ± ± ± ± GM 8.87± ± ± ± ± ± ± CK 8.51± ± ± ± ± ± ± NM 8.81± ± ± ± ± ± ± GM 8.90± ± ± ± ± ± ± bacteria-day 90 CK 9.12± ± ± ± ± ± ± NM 9.20± ± ± ± ± ± ± GM 9.15± ± ± ± ± ± ± CK 9.18± ± ± ± ± ± ± NM 9.08± ± ± ± ± ± ± GM 9.06± ± ± ± ± ± ± Duodenum CK 6.24± ± ± ± ± ± ± NM 6.42± ± ± ± ± ± ± GM 6.84± ± ± ± ± ± ± CK 6.53± ± ± ± ± ± ± NM 6.76± ± ± ± ± ± ± GM 6.53± ± ± ± ± ± ± Ileum CK 7.70± ± ± ± ± ± ± NM 7.58± ± ± ± ± ± ± GM 7.77± ± ± ± ± ± ± CK 7.61± ± ± ± ± ± ± NM 7.52± ± ± ± ± ± ± GM 7.86± ± ± ± ± ± ± Caecum CK 8.76± ± ± ± ± ± ± NM 9.31±0.49 * 9.16± ± ± ±0.57 * 7.15± ± GM 9.36±0.34 * 9.19± ± ± ±0.51 * 6.98± ± CK 8.61± ± ± ± ± ± ± NM 9.17±0.57 * 9.28± ± ± ±0.58 * 6.99± ± GM 9.20±0.38 * 9.16± ± ± ±0.6 * ± ± Data are shown as log10 cfu g-1 ± SD. *:Statistically significant differences from the CK group (p < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group.

6 Table S4 Feces Cecum Wet weight (g/d) Water content % ph Wet weight (g) ph CK 5.05± ± ± ± ±0.14 NM 5.36± ± ± ± ±0.09 * GM 5.28± ± ± ± ±0.11 * CK 4.27± ± ± ± ±0.15 NM 4.31± ± ± ± ±0.18 * GM 4.44± ± ± ± ±0.18 * Values are means ± SD for 6 rats per dietary group. *:Statistically significant difference from the CK group (p < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group.

7 Table S5 Cecal content Male Female CK NM GM CK NM GM Acetate 33.96± ±5.62 * 38.99± ± ± ±5.69 Propionate 10.76± ± ± ± ± ±2.82 Butyrate 9.15± ± ± ± ± ±1.46 Total SCFA 53.62± ± ± ± ± ±5.78 Fecal content Acetate 19.21± ± ± ± ± ±2.91 Propionate 5.38± ± ± ± ± ±1.89 Butyrate 3.21± ± ± ± ± ±1.22 Total SCFA 27.8± ± ± ± ± ±5.42 Values are mean ± SD of eight rats from each group. *:Statistically significant difference from the CK group (p < 0.05) CK: AIN93 G group; NM: non-genetically modified group; GM: genetically modified group; SCFAs: short-chain fatty acids.

8 Table S6

9 Target bacteria Amplicon size (bp) Annealing temperature ( C) Sequence (5 3 ) Reference Lactobacillus group Bifidobacterium genus Escherichia coli subgroup Enterococcus genus Clostridium perfringens Bacteroides Prevotella group Total bacteria GC-PRBA338F& 518R PRBA338F & 518R β-actin ZO occludin Bar Bar Cry2A Cry2A Cry2A Cry2A Cry2A Cry2A Flank sequence Flank sequence NOS s SPS Ubiquitin Chloroplast F: AGCAGTAGGGAATCTTCCA R: CACCGCTACACATGGAG F: GGGTGGTAATGCCGGATG R: TAAGCCATGGACTTTCACACC F: GTTAATACCTTTGCTCATTGA R: ACCAGGGTATCTAATCCTGTT F: CCCTTATTGTTAGTTGCCATCATT R: ACTCGTTGTACTTCCCATTGT F: ATGCAAGTCGAGCGAKG R: TATGCGGTATTAATCTYCCTTT F: GAAGGTCCCCCACATTG R: CAATCGGAGTTCTTCGTG F: TCCTACGGGAGGCAGCAGT R: GGACTACCAGGGTATCTAATCCTGTT F: CGCCCGCCGCGCGCGGCGGGCGGGGCGGGG GCACGGGGGGACTCCTACGGGAGGCAGCAG R: ATTACCGCGGCTGCTGG- F: ACTCCTACGGGAGGCAGCAG R: ATTACCGCGGCTGCTGG- F: CCCATCTATGAGGGTTACGC R: TTTAATGTCACGCACGATTTC F: GAGGCTTCAGAACGAGGCTATTT R: CATGTCGGAGAGTAGAGGTTCGA F: GCCTATGGAACGGGCATCTT R: GCCAGCAGGAAACCCTTTG- F: GCACCATCGTCAACCACTACA R: GTCCACTCCTGCGGTTCCT F: ACTGGGCTCCACGCTCTACA R: ATGACAGCGACCACGCTCTT F: ACGGACCACAGCCTCTACGT R: TGAGGCTACCCACCTTCTTCA F: ACCCTCGCTCGTGTCAATG R: GTTGTCCACCTGTTGATTGAACTC F: CATGCTTGAGTTCCGCACAT R: TGGTACTTGAAGAGGCTCCAGAT F: AGTTCCAGATCCAAGGCTACCA R: GATGAAGCTCAGGTGCATGTTG F: ACAGCGCCAGGGTGAACTAC R: GTTGAAGTTGTGGTTGAGATTGGT F: TGGCCCCCGAAGACTACA R: GAAAGTGCGGGTTTGATTGTTC F: CGCAATGTGTTATTAAGTTGTCTAAGC R: GAGATATGATGATACTCCGTTTGCA F: TGCAATGCAAACGGAGTATCAT R: CGTATCTCGTCAATTCCAATTCAG F: GAATCCTGTTGCCGGTCTTG-3 R: TTATCCTAGTTTGCGCGCTA-3 F: GCTCCTACAAATGCCATCA-3 R: GATAGTGGGATTGTGCGTCA-3 F: TTGCGCCTGAACGGATAT-3 R: GGAGAAGCACTGGACGAGG-3 F: AACACTGGCAAGTTAGCAAT-3 R: CCGTAATAAATAGACACCC-3 F: CGAAATCGGTAGACGCTACG-3 R: TTCCATTGAGCTTCTGCA CCT-3 Walter et al. 2001; Heilig et al Bartosch et al Malinen et al Rinttila et al Rinttila et al Bartosch et al Nadkarni et al Lee, et al.2005 Lee, et al.2005

11 Supplementary Figures Fig Sl

12 Fig S2

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5- CACGAAACTACCTTCAACTCC-3 beta actin-r 5- CATACTCCTGCTTGCTGATC-3 GAPDH-F GAPDH-R Table S1 Primers used in this work LncPPARδ-F 5- AGGATCCCAAGGCAGAAGTT -3 LncPPARδ-R 5- GAATAGAAAGGCGGGAAAGG -3 PPARδ-F 5- CACCCAGTGTCCTTCCATCT -3 PPARδ-R 5- CAGAATTGGGTGTGCTGCTT -3 ADRP-F 5- ACAACCGAGTGTGGTGACTC