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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 袁建琴等 / 转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析 May 25, 2016, 32(5): Chin J Biotech, All rights reserved 食品生物技术 657 SD 袁建琴 1, 常泓 1, 赵江河 1, 史宗勇 1, 王俊东 ,,,. SD., 2016, 32(5): Yuan JQ, Chang H, Zhao JH, et al. Analysis of exogenous gene and protein digestion and absorption of SD rats (Rattus norvegicus) fed roundup ready soybean meal. Chin J Biotech, 2016, 32(5): 摘要 : 为了评估转基因抗草甘膦除草剂大豆的食用安全性, 以 20% 的比例将转基因抗草甘膦除草剂大豆 GTS 和其亲本非转基因大豆 A5403 豆粕分别添加到基础饲料中喂养两代 Sprague-Dawley (SD) 大鼠, 采用定性定量 PCR 和 ELISA 方法检测转基因大豆成分相关基因和蛋白在长期饲喂的大鼠体内代谢残留状况结果表明, 大鼠喂养转基因大豆豆粕后, 除了大鼠肠粪和盲肠内容物检测到有转基因成分的残留, 肠道菌群和实质脏器均未发现相关基因和蛋白结果提示, 长期饲喂转基因抗草甘膦除草剂大豆 GTS 与亲本 A5403 大豆豆粕对 SD 大鼠具有同样的食用安全性 : 抗草甘膦除草剂, 转基因大豆 GTS40-3-2,SD 大鼠, 外源基因和蛋白, 消化吸收 Received: December 13, 2015; Accepted: March 16, 2016 Supported by: Key Projects in the National Science and Technology Pillar Program during the Twelfth Five-Year Plan Period of China (No. 2012BAD12B06-2), Science and Technology Key Program of Shanxi Province (No ), Natural Science Foundation of Shanxi Province (No ), Higher School Teaching Reform Project of Shanxi Province (No. J ). Corresponding author: Jundong Wang. Tel: ; fax: ; wangjd53@outlook.com (No. 2012BAD12B06-2) (No ) (No ) (No. J )

2 658 ISSN CN /Q Chin J Biotech May 25, 2016 Vol.32 No.5 Analysis of exogenous gene and protein digestion and absorption of SD rats (Rattus norvegicus) fed roundup ready soybean meal Jianqin Yuan 1, Hong Chang 1, Jianghe Zhao 1, Zongyong Shi 1, and Jundong Wang 2 1 College of Life Science, Shanxi Agricultural University, Taigu , Shanxi, China 2 College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu , Shanxi, China Abstract: Metabolism and deposition of exogenous gene and protein from transgenic glyphosate herbicide-tolerant soybean meal in SD rats were studied in the experiment. The transgenic soybean GTS meal and its non-transgenic counterpart (parent A5403) were fed to the generation and the second generation Sprague-Dawley (SD) rats (Rattus norvegicus). The study added the genetically modified (GM) soybean meal and its non-transgenic control soybean meal (parent A5403) in a ratio of 20% respectively to the feeds. By using qualitative, quantitative PCR and ELISA methods to detect transgenic soybean residues of metabolism ingredients in rats, the safety and influence of GM soybean were evaluated. The results showed that the intestinal fecal and cecum contents of rats were detected with residues of GM ingredients, intestinal flora and organs were not found related genes and protein. These results indicated that transgenic glyphosate herbicide-tolerant soybean GTS meal was as safe as its non-gm soybean meal in long-term feeding study. Keywords: glyphosate-resistance herbicide, GM soybeans GTS40-3-2, SD rats, exogenous gene and protein, digestion and absorption [1] [2-6] [1-3] [7-9] 90 d [5-12] 90 d [13] 20% GTS [6,9,14] A5403 Sprague Dawley (SD) PCR

3 袁建琴等 / 转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析 659 ELISA GTS SD 1 材料与方法 1.1 材料 g SD 5 d 4 ( ) GMF (8 ) GMM (4 ) non-gmf (8 ) non-gmm (4 ) 12 h 12 h 23 ±1 55%±15% , / 21 d ( ) 3 ( ) ( 5 ) g 4 ( 10 ) d % GTS (IRMM) (Catalog Number ERM-BF410GK) ( GTS ( ) A5403) ( ) PCR ( 20%) [14] DNA ( ) TransTaq-T DNA Polymerase TaKaRa MiniBest Universal Genomic DNA Extraction kit ver 5.0 Premix Ex Taq TM (Probe qpcr) DL2 000 DNA Marker TaKaRa ( ) ELISA CP4 EPSPS Roundup Agdia PCR TaqMan Escherichia coli ATCC Lactobacillus acidophilus ATCC PCR GTS PCR 1 [15-16] S rrna zx338f rg7f (zx338f rg7f ) [17-19] cjb@im.ac.cn

4 660 ISSN CN /Q Chin J Biotech May 25, 2016 Vol.32 No.5 表 1 定性 PCR 引物序列 Table 1 The qualitative PCR primers sequences Primer name Orientation Primer sequence (5 3 ) Amplicon size (bp) lectin GCCCTCTACTCCACCCCCATCC GCCCATCTGCAAGCCTTTTTGTG 118 CaMV35S GCTCCTACAAATGCCATCATTGC GATAGTGGGATTGTGCGTCATCCC 195 nos GAATCCTGTTGCCGGTCTTG TTATCCTAGTTTGCGCGCTA 180 cp4-epsps CCTTCATGTTCGGCGGTCTCG GCGTCATGATCGGCTCGATG s-ctp4 TGATGTGATATCTCCACTGACG TGTATCCCTTGAGCCATGTTGT 171 表 2 检测 SD 大鼠肠道菌群引物 Table 2 The primers of detecting SD rat intestinal bacteria Primer name Orientation Primer sequence (5 3 ) Amplicon size (bp) zx338f ACTCCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG 171 rg7f AGAGTTTGATYMTGGCTCAG CTCAAAACTAAACAAAGTTTC PCR [20] 方法 SD 4 5 g h LBS h LBS (1 4 GMM GMF non-gmm non-gmf ) (5 8 GMM GMF non-gmm non GMF ) DNA DNA TransTaq-T DNA Polymerase CP4-EPSPS ELISA ( ) 4 ELISA CP4 EPSPS Roundup CP4-EPSPS

5 袁建琴等 / 转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析 661 表 3 实时荧光定量 PCR 引物序列 Table 3 Real-time PCR primer sequences Primer name Orientation Primer sequence (5 3 ) CAT TTG GAG AGG ACA CGC TGA Amplicon size (bp) GTS GAG CCA TGT TGT TAA TTT GTG CC 74 Probe FAM-CAA GCT GAC TCT AGC AGA TCT TTC-TAMRA CCA GCT TCG CCG CTT CCT TC lectin GAA GGC AAG CCC ATC TGC AAG CC 74 Probe FAM-CTT CAC CTT CTA TGC CCC TGA CAC-TAMRA 表 4 样品编号 Table 4 Sample serial number Serial number The sample Serial number The sample 1 Spleen 7 Kidneys 2 Live 8 Lung 3 Heart 9 Testis 4 Stomach 10 Brain 5 Intestinal fecal 11 Ovaries 6 Cecum contents 12 Adrenal gland SD PCR 4 SD DNA TaKaRa MiniBest Universal Genomic DNA Extraction kit ver 5.0 TransTaq-T DNA Polymerase SD PCR DNA Premix Ex Taq TM (Probe qpcr) lectin GTS GTS GTS 结果与分析 2.1 SD 大鼠肠道菌群转基因成分相关基因的检测 SD DNA DNA μg/ml DNA (OD 260/ OD 260/ ) GTS SD DNA DNA PCR DNA rg7f zx338f zx338f ATCC rg7f ATCC A kb rg7f 5 8 1B 8 DNA 171 bp zx338f 1A B 8 2 DNA DNA DNA cjb@im.ac.cn

662 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech May 25, 2016 Vol.32 No.5 35s-ctp4 cp4-epsps ( 5) 2.1.3 PCR 8 DNA lectin GTS40-3-2 PCR ( 6) 2.

6 7 8 lectin CaMV35S 图 1 rg7f 和 zx338f 基因 PCR 扩增产物电泳图 Fig. 1 The electrophoretogram of PCR products of rg7f and zx338f genes.

6 662 ISSN CN /Q Chin J Biotech May 25, 2016 Vol.32 No.5 35s-ctp4 cp4-epsps ( 5) PCR 8 DNA lectin GTS PCR ( 6) 2.2 SD 大鼠脏器组织 ELISA 检测结果 ELISA CP4 EPSPS Roundup ( 7) 650 nm 表 5 转基因大豆 GTS 内外源基因检测电泳结果 Table 5 Electrophoresis results of GM soybeans GTS internal and external source genes Genes lectin CaMV35S 图 1 rg7f 和 zx338f 基因 PCR 扩增产物电泳图 Fig. 1 The electrophoretogram of PCR products of rg7f and zx338f genes. The electrophoregram A and B respectively on behalf of detecting rg7f and zx338f genes. 1, 2, 9, 10 lanes: 1, 5 samples (GMM group); 3, 4, 11, 12 lanes: 2, 6 samples (GMF group); 5, 6, 17, 18 lanes: 3, 7 samples (non-gmm group); 7, 8, 19, 20 lanes: 4, 8 samples (non-gmf)); 13, 14, 29 and 30 lanes: positive control; 15, 16, 31 and 32 lanes: negative control; M: DL2 000 DNA marker (2 000 bp, bp, 750 bp, 500 bp, 250 bp, 100 bp) GTS PCR GTS SD DNA 8 GTS lectin CaMV35S nos nos 35s-ctp4 cp4-epsps Note: indicated the negative results. 表 6 肠道菌群实时荧光定量 PCR 检测结果 Table 6 Real-time PCR detection results of intestinal flora Genes lectin GTS Note: indicated the negative results. 表 7 结果分析参考对照 Table 7 Result analyse contrast OD value The results >0.3 Positive <0.1 Negative Not sure, need more analysis

袁建琴等/转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析孔的 OD 值阳性质控物吸光度值为 0.629±0.023 与定量 PCR 检测大于阳性标准 0.3 呈阳性阴性质控物吸光度值 2.3.1 SD 大鼠脏器组织 DNA 定性 PCR 检测为 0.069±0.012 小于阴性标准 0.

7 袁建琴等/转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析孔的 OD 值阳性质控物吸光度值为 0.629±0.023 与定量 PCR 检测大于阳性标准 0.3 呈阳性阴性质控物吸光度值 SD 大鼠脏器组织 DNA 定性 PCR 检测为 0.069±0.012 小于阴性标准 0.1 试验结果可对提取的 12 个 SD 大鼠脏器 DNA 进行转基靠可进行样品数据的判定最终样品 8 号和 9 因抗草甘膦除草剂大豆 GTS 内外源相关号 (肠粪和盲肠内容物) 数值分别为 0.237±0.010 基因定性 PCR 检测每个样品 2 次重复除了和 0.238±0.030 位于 0.1 和 0.3 之间结果可疑大鼠肠粪和盲肠内容物扩增出了 118 bp 的大豆进行以下相关基因检测验证其余样品吸光度值内标准 lectin 基因 195 bp 的 CaMV35S 启动子均小于阴性标准 0.1 未检出转基因大豆外源蛋白 CP4-EPSPS 残留 2.3 SD 大鼠脏器组织转基因成分相关基因检测对提取的 SD 大鼠脏器组织 DNA 进行定性 180 bp 的 nos 终止子 498 bp 的 cp4-epsps 和 171 bp 的 35s-ctp4 外源基因外其他 SD 大鼠实质脏器组织均未扩增出相应条带 (图 2) 对上述肠粪和盲肠内容物的 PCR 产物测序 5 个基因的序列与文献报道序列一致 [15 16] 图 2 Lectin CaMV35S nos cp4-epsps 和 35s-ctp4 扩增产物电泳图 Fig. 2 The electrophoretogram of PCR products of lectin, CaMV35S, nos, cp4-epsps and 35s-ctp4 genes. The electrophoregram A, B, C, D and E respectively on behalf of detecting lectin, CaMV35S, nos, cp4-epsps and 35s-ctp4 genes; 1-12 and lanes: samples (1 and 2 lanes: spleen; 3 and 4 lanes: live; 5 and 6 lanes: heart; 7 and 8 lanes: stomach; 9 and 10 lanes: intestinal fecal; 11 and 12 lanes: cecum contents; 17 and 18 lanes: kidneys; 19 and 20 lanes: lung; 21 and 22 lanes: testis; 23 and 24 lanes: brain; 25 and 26 lanes: ovaries; 27 and 28 lanes: adrenal gland); 13, 14, 29 and 30 lanes: positive control; 15, 31 lanes: negative control; 16, 32 lanes: blank; M: DL2 000 DNA marker (2 000 bp, bp, 750 bp, 500 bp, 250 bp, 100 bp) cjb@im.ac.cn 663

8 664 ISSN CN /Q Chin J Biotech May 25, 2016 Vol.32 No SD DNA PCR 12 SD DNA lectin PCR lectin ( 3) 3 lectin 97.4% R > (> 3.6 < 3.1) DNA lectin SD DNA lectin C t /2 μl C t /2 μl SD DNA GTS PCR GTS GTS % R > (> 3.6 < 3.1) DNA GTS SD DNA GTS 图 3 Lectin 基因标准曲线图和扩增曲线图 Fig. 3 The standard and amplification curves of lectin gene. The curve A and B respectively on behalf of the standard and amplification curves of lectin gene. 图 4 GTS 基因标准曲线图和扩增曲线图 Fig. 4 The standard and amplification curves of GTS gene. The curve A and B respectively on behalf of the standard and amplification curves of GTS gene.

9 袁建琴等 / 转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析 665 Ct /2 μl Ct /2 μl 10 SD SD GTS n C= GTS (n GTS n lectin GTS n lectin lectin ) SD GTS ( ) 55.4% GTS ( ) 55.6% 3 讨论 [7] [21-22] [17-19,23] DNA [17-19] PCR DNA SD 2004 Alexander PCR PCR cp4 epsps DNA, ph DNA DNA [24] 2008 Bakke-McKellep 6% [25] 2009 Sissener [26] 2011 [8] 2012 Chukwudebe CV d CV127 [27] ELISA CP4-EPSPS CP4-EPSPS GTS CP4-EPSPS ( OD ±0.010 OD ±0.030) 2 OD PCR GTS SD PCR lectin cjb@im.ac.cn

10 666 ISSN CN /Q Chin J Biotech May 25, 2016 Vol.32 No.5 CaMV35S nos 35s-ctp4 cp4-epsps 5 ( PCR cp4-epsps ELISA CP4-EPSPS ) PCR lectin GTS (GTS % 55.6%) GTS 结论 20% GTS SD PCR ELISA GTS A5403 REFERENCES [1] Huang DF. Review of transgenic crop breeding in China. Chin J Biotech, 2015, 31(6): (in Chinese). 昉.., 2015, 31(6): [2] Guo JC. Development of new DNA-based methods for analysis of genetically modified plants and their derived products [D]. Shanghai: Shanghai Jiao Tong University, 2011 (in Chinese).. [D]. :, [3] Tan JZ. The feed safety assessment of glyphosate-tolerant soybean meal in broilers [D]. Beijing: Chinese Academy of Agriculture Sciences, 2011 (in Chinese).. [D]. :, [4] James C. Global status of commercialized Biotech/GM crops: China Biotech, 2015, 35(1): 1 14 (in Chinese). James C /., 2015, 35(1): [5] Zhu YZ, Yin JD, Li DF, et al. Study on metabolism of exogenous DNA from transgenic soybean meal in grower pigs. Acta Vet Zootech Sin, 2005, 36(10): (in Chinese).,,,. DNA., 2005, 36(10): [6] Zhao ZH, Yang LT, Ai XJ, et al. Analysis of the influence on physiological metabolism and genetic horizontal transformation of rats fed roundup ready soybean meal. J Nanjing Agr Univ, 2006, 29(1): (in Chinese).,,,.., 2006, 29(1): [7] Lu CB, Zhang W, Liu B. Effects of transgenic soybean feed on proliferation of spleen lymphocyte in male mice. Soy Sci, 2012, 31(2): (in Chinese).,,.., 2012, 31(2): [8] Tan JZ, Liu SS, Sun Z, et al. Effects of glyphosate-tolerant soybean meal on intestinal mucosal immunity in broilers. Chin J Anim Nutr, 2011, 23(5): (in Chinese).,,,.., 2011, 23(5): [9] Lu CB, Cai J, Qi QL, et al. Effects of glyphosate-resistant transgenic soybean on male

11 袁建琴等 / 转基因大豆豆粕外源基因和蛋白在 SD 大鼠体内消化吸收分析 667 mice testis. J Jinan Univ: Nat Sci Med Ed, 2014, 35(3): (in Chinese).,,,.. :, 2014, 35(3): [10] Zhu YX, He XY, Luo YB, et al. A 90-day feeding study of glyphosate-tolerant maize with the G2-aroA gene in Sprague-Dawley rats. Food Chem Toxicol, 2013, 51: [11] Demerlis CC, Schoneker DR, Borzelleca JF. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem Toxicol, 2014, 70: [12] Chen DL, Zhang SM, Zou SY, et al. A 90-day subchronic feeding study of transgenic glyphosate herbicide-resistant maize CC-2 in Sprague-Dawley rats (Rattus norvegicus). J Agr Biotech, 2013, 21(12): (in Chinese).,,,. CC-2 SD 90., 2013, 21(12): [13] Ministry of Agriculture of the People's Republic of China. NY/T Safety assessment of genetically modified plant and derived products 90-day feeding test in rats. Beijing: China Standards Press, 2006 (in Chinese).. NY/T :, [14] General Administration of quality supervision, inspection and Quarantine of the People's Republic of China, China National Standardization Management Committee. GB Laboratory animals-nutrients for formula feeds. Beijing: China Standards Press, 2011 (in Chinese).,. GB :, [15] Ministry of Agriculture of the People's Republic of China. Chinese Standard Agriculture Department Public Announcement No detection of genetically modified plants and derived products. Qualitative PCR method for the regulatory elements CaMV 35S promoter, FMV 35S promoter, NOS promoter, NOS terminator and CaMV 35S terminator. Beijing: China Agriculture Press (in Chinese) CaMV 35S FMV 35S NOS NOS CaMV 35S PCR. :, [16] Ministry of Agriculture of the People's Republic of China. Chinese Standard Agriculture Department Public Announcement No detection of genetically modified plants and derived products. Qualitative PCR method for herbicide-tolerant soybean GTS and its derivates. Beijing: China Agriculture Press, 2013 (in Chinese) GTS PCR. :, [17] Li LT, Xu WT, Guo X, et al. An effective method for genomic DNA extraction from rat intestinal microflora. J Chin Cereals Oils Assoc, 2010, 25(1): (in Chinese).,,,. DNA., 2010, 25(1): [18] Gong Q, Guan DM, Wang YB, et al. Methods extracted Escherichia coli genomic DNA using the fast ways. Mar Environ Sci, 2005, 24(4): (in Chinese).,,,. DNA., 2005, 24(4): [19] Zhang SL, Liu YX, Zhang HQ, et al. Development of a genus-specific T-RFLP technique for differentiating Lactobacillus community. Microbiol China, 2012, 39(8): (in Chinese).,,,. T-RFLP., cjb@im.ac.cn

12 668 ISSN CN /Q Chin J Biotech May 25, 2016 Vol.32 No , 39(8): [20] General Administration of Quality Supervision, Inspection and Quarantine. GB/T detection of genetically modified organisms and derived products-quantitative nucleic acid based methods. Beijing: China Standards Press, 2004 (in Chinese).. GB/T PCR. :, [21] Nicholson JK, Holmes E, Wilson ID. Gut microorganisms, mammalian metabolism and personalized health care. Nat Rev Microbiol, 2005, 3(5): [22] Ji X, Zheng WJ, Zou XT, et al. Analysis of antibiotic and copper resistance of Escherichia coli isolated from feces of sows and piglets before and after weaning. Acta Vet Zootech Sin, 2015, 46(3): (in Chinese).,,,.., 2015, 46(3): [23] Zheng G, Chen JR, Hu BW, et al. Genomic DNA extraction from rat faecal and intestinal microflora based on DGGE analysis. Food Sci, 2011, 32(17): (in Chinese).,,,. DGGE DNA., 2011, 32(17): [24] Alexander TW, Sharma R, Deng MY, et al. Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready canola in the intestinal, ruminal, and fecal contents of sheep. J Biotechnol, 2004, 112(3): [25] Bakke-McKellep AM, Sanden M, Danieli A, et al. Atlantic salmon (Salmo salar L.) parr fed genetically modified soybeans and maize: histological, digestive, metabolic, and immunological investigations. Res Vet Sci, 2008, 84(3): [26] Sissener NH, Bakke AM, Gu J, et al. An assessment of organ and intestinal histomorphology and cellular stress response in Atlantic salmon (Salmo salar L.) fed genetically modified Roundup Ready soy. Aquaculture, 2009, 298(1/2): [27] Chukwudebe A, Privalle L, Reed A, et al. Health and nutritional status of Wistar rats following subchronic exposure to CV127 soybeans. Food Chem Toxicol, 2012, 50(3/4): ( 本文责编郝丽芳 )

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇