Hostname: page-component-cd9895bd7-p9bg8 Total loading time: 0 Render date: 2024-12-23T09:00:33.540Z Has data issue: false hasContentIssue false
  • English
  • Français

Relative stability of transgene DNA fragments from GM rapeseed in mixed ruminal cultures

Published online by Cambridge University Press:  09 March 2007

Ranjana Sharma ,
Trevor W. Alexander ,
S. Jacob John ,
Robert J. Forster  and
Tim A. McAllister
Ranjana Sharma
Affiliation:
Agriculture and Agri-Food Canada Research Center, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1
Trevor W. Alexander
Affiliation:
Agriculture and Agri-Food Canada Research Center, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, CanadaT6G 2P5
S. Jacob John
Affiliation:
Aquaculture Centre for Excellence, Lethbridge Community College, Lethbridge, Alberta, Canada T1K 1L6
Robert J. Forster
Affiliation:
Agriculture and Agri-Food Canada Research Center, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1
Tim A. McAllister*
Affiliation:
Agriculture and Agri-Food Canada Research Center, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1
*
*Corresponding author: Dr Tim A. McAllister, fax +1 403 3823156, email [email protected]
Rights & Permissions [Opens in a new window]

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

The use of transgenic crops as feeds for ruminant animals has prompted study of the possible uptake of transgene fragments by ruminal micro-organisms and/or intestinal absorption of fragments surviving passage through the rumen. The persistence in buffered ruminal contents of seven different recombinant DNA fragments from GM rapeseed expressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene was tracked using PCR. Parental and transgenic (i.e. glyphosphate-tolerant; Roundup Ready®, Monsanto Company, St Louis, MO, USA) rapeseed were incubated for 0, 2, 4, 8, 12, 24 and 48 h as whole seeds, cracked seeds, rapeseed meal, and as pelleted, barley-based diets containing 65 g rapeseed meal/kg. The seven transgene fragments ranged from 179 to 527 bp and spanned the entire 1363 bp EPSPS transgene. A 180 bp ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fragment and a 466 bp 16S rDNA fragment were used as controls for endogenous rapeseed DNA and bacterial DNA respectively. The limit of detection of the PCR assay, established using negative controls spiked with known quantities of DNA, was 12·5 pg. Production of gas and NH3 was monitored throughout the incubation and confirmed active in vitro fermentation. Bacterial DNA was detected in all sample types at all time points. Persistence patterns of endogenous (Rubisco) and recombinant (EPSPS) rapeseed DNA were inversely related to substrate digestibility (amplifiable for 48, 8 and 4 h in whole or cracked seeds, meal and diets respectively), but did not differ between parental and GM rapeseed, nor among fragments. Detection of fragments was representative of persistence of the whole transgene. No EPSPS fragments were amplifiable in microbial DNA, suggesting that transformation had not occurred during the 48 h incubation. Uptake of transgenic DNA fragments by ruminal bacteria is probably precluded or time-limited by rapid degradation of plant DNA upon plant cell lysis.

Keywords

Genetically modifiedEPSPS gene fragmentsRoundup Ready®rapeseedRubiscoForeign DNA stability
Type
Research Article
Information
British Journal of Nutrition , Volume 91 , Issue 5 , May 2004 , pp. 673 - 681
Copyright
Copyright © The Nutrition Society 2004

References

Alexander, TW, Sharma, R, Okine, EK, Dixon, WT, Forster, RJ, Stanford, K & McAllister, TA (2002) Impact of feed processing and mixed ruminal culture on the fate of recombinant EPSP synthase and endogenous canola plant DNA. FEMS Microbiol Lett 214, 263–269.Google Scholar
Broderick, GA & Kang, JH (1980) Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J Dairy Sci 63, 64–75.CrossRefGoogle ScholarPubMed
Canola Council of Canada (2001), Impact of Transgenic Canola on Growers, Industry and Environment. www.canola-council. org/manual/GMO/gmo_main.htmGoogle Scholar
Cheng, K-JJ & McAllister, TA (1997) Compartmentation in the rumen. In The Rumen Microbial Ecosystem, 2nd ed. pp. 492–522 [Hobson, PN and Stewart, CS, editors]. London: Blackie Academic and Professional.CrossRefGoogle Scholar
Duggan, PS, Chambers, PA, Heritage, J & Forbes, JM (2000) Survival of free DNA encoding antibiotic resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent. FEMS Microbiol Lett 191, 71–77.Google Scholar
Duggan, PS, Chambers, PA, Heritage, J & Forbes, JM (2003) Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. Br J Nutr 89, 159–166.CrossRefGoogle ScholarPubMed
Einspanier, R, Klotz, A, Kraft, J, Aulrich, K, Poser, R, Schwagele, F, Jahreis, G & Flachowsky, G (2001) The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material. Eur Food Res Technol 212, 129–134.Google Scholar
Fedorak, PM & Hrudey, SE (1983) A simple apparatus for measuring gas production by methanogenic cultures in serum bottles. Environ Technol Lett 4, 425–432.CrossRefGoogle Scholar
Garcia-Vallve, S, Romeu, A & Palau, J (2000) Horizontal gene transfer of glycosyl hydrolases of the rumen fungi. Mol Biol Evol 17, 352–361.Google Scholar
Health Canada (1999) Novel Food Information – Food Biotechnology. Glyphosate Tolerant Canola, GT200. http://www.hc-sc.gc.ca/food-aliment/mh-dm/ofb-bba/nfi-ani/e_ofb-097-325-a.htmlGoogle Scholar
Hohlweg, U & Doerfler, W (2001) On the fate of plant or other foreign genes upon the uptake in food or after intramuscular injection in mice. Mol Genet Genomics 265, 225–233.Google Scholar
Kleter, GA & Kuiper, HA (2002) Considerations for the assessment of the safety of genetically modified animals used for human food or animal feed. Livest Prod Sci 74, 275–285.CrossRefGoogle Scholar
McAllan, AB & Smith, RH (1973) Degradation of nucleic acids in the rumen. Br J Nutr 29, 331–345.CrossRefGoogle ScholarPubMed
McAllister, TA, Bae, HD, Jones, GA & Cheng, K-J (1994) Microbial attachment and feed digestion in the rumen. J Anim Sci 72, 3004–3018.CrossRefGoogle ScholarPubMed
Martens, MA (2000) Safety evaluation of genetically modified foods. Int Arch Occup Environ Health 73, Suppl. 1, S14–S18.Google Scholar
Menke, KH, Raab, L, Salewski, A, Steinglass, H, Fritz, D & Schneider, W (1979) The estimation of the digestibility and metabolizable energy content of ruminant feeding stuffs from the gas production when they are incubated with ruminal culture on the fate of recombinant EPSP synthase and endogenous canola plant DNA. FEMS Microbiol Lett 214, 222–263.Google Scholar
Morrison, M (1996) Do ruminal bacteria exchange genetic material. J Dairy Sci 79, 1476–1486.Google Scholar
Nadkarni, MA, Martin, FE, Jacques, NA & Hunter, N (2002) Determination of bacterial load by real-time PCR using a broad range (universal) probe and primer set. Microbiol SGM 148, 257–266.Google Scholar
Nikolich, MP, Hong, G, Shoemaker, NB & Salyers, AA (1994) Evidence for natural horizontal transfer of tetQ between bacteria that normally colonize humans and bacteria that normally colonize livestock. Appl Environ Microbiol 60, 3255–3260.Google Scholar
Organization of Economic Cooperation and Development (1993) Food safety and biotechnology: concepts and principles. In Safety Evaluation of Foods Derived by Modern Biotechnology: Concepts and Principles, pp. 10–13 [Environment Directorate, OECD, editors]. Paris: OECD http://www.oecd.org/dataoecd/57/3/1946129.pdfGoogle Scholar
Phipps, RH, Beever, DE & Humphries, DJ (2002) Detection of transgenic DNA in milk from cows receiving herbicide tolerant (CP4EPSPS) soyabean meal. Livest Prod Sci 74, 269–273.Google Scholar
Ruiz, TR, Andres, S & Smith, GB (2000) Identification and characterization of nuclease activities in anaerobic environmental samples. Can J Microbiol 46, 736–740.Google Scholar
Sambrook, J, Fritsch, EF & Maniatis, T (1989) Gel electrophoresis of DNA. In Molecular Cloning: A Laboratory Manual, 2nd ed. pp. 6.9–6.13 [Ford, N and Nolan, C, editors]. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.Google Scholar
Smith, RH & McAllan, AB (1970) Nucleic acid metabolism in the ruminant. Br J Nutr 24, 545–556.CrossRefGoogle ScholarPubMed
Statistics Canada (2003) Livestock Feed Requirements Study. Canada and Provinces 1999, 2000, 2001. Catalogue no. 23-501-XIE. Ottawa, Canada: Statistics Canada.Google Scholar
Wallace, RJ, Onodera, R & Cotta, MA (1997) Metabolism of nitrogen-containing compounds. In The Rumen Microbial Ecosystem, pp. 283–328 [Hobson, PN and Stewart, CS, editors]. New York: Blackie Academic & Professional.Google Scholar
Wang, Y, McAllister, TA, Zobell, DR, Pickard, MD, Rode, LM, Mir, Z & Cheng, K-J (1997) The effect of micronization of full-fat canola seed on digestion in the rumen and total tract of dairy cows. Can J Anim Sci 77, 431–440.Google Scholar