title: Relative stability of transgene DNA fragments from GM rapeseed in mixed ruminal cultures.
abstract: 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.
authors.affiliation: Agriculture and Agri-Food Canada Research Center, PO Box 3000, Lethbridge, Alberta Canada T1J 4B1.
authors.forename: Ranjana
authors.initials: R
authors.lastname: Sharma
authors.forename: Trevor W
authors.initials: TW
authors.lastname: Alexander
authors.forename: S Jacob
authors.initials: SJ
authors.lastname: John
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Forster
authors.forename: Tim A
authors.initials: TA
authors.lastname: McAllister
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D029741:Brassica rapa / Q000235:genetics*; D002417:Cattle; D053938:DNA Fragmentation / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics; D004063:Digestion / Q000502:physiology; D008297:Male; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism; D012417:Rumen / Q000382:microbiology / Q000502:physiology*; D019076:Transgenes / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; D004274:DNA, Recombinant; D000641:Ammonia; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D012273:Ribulose-Bisphosphate Carboxylase