In situ studies on the time-dependent degradation of recombinant corn DNA and protein in the bovine rumen.
An in situ technique was adopted to investigate the time-dependent ruminal degradation of chloroplast compared with recombinant DNA of Bt176 corn using conventional and quantitative PCR assays. In parallel, the Cry1Ab protein content and fragment sizes were determined by ELISA and immunoblotting techniques. Triplicate nylon bags filled with 5 g of each substrate (whole-plant isogenic, whole-plant transgenic, ensiled isogenic, and ensiled transgenic corn) were positioned within the rumen of 5 rumen-cannulated, nonlactating cows and incubated for 2, 4, 8, 16, 24, and 48 h. To investigate the DNA degradation process, PCR assays were developed to detect fragments of the endogenous highly abundant rubisco gene (173, 896, 1,197, and 1,753 bp) and of the recombinant cry1Ab gene (211, 420, 727, and 1,423 bp). Short fragments of rubisco (<431 bp) and cry1Ab DNA (211 bp) were amplifiable in whole-plant and ensiled corn samples incubated in the rumen for 48 h, whereas the traceability of larger fragments depended on previous processing of the sample (whole-plant or ensiled corn), the length of the target sequence, and concomitantly on the length of time incubated in the rumen. Quantification of rubisco and cry1Ab gene fragments applying real-time PCR assays revealed degradation to <20% of initial 0-h values within 2 h and <0.5% after 48 h of ruminal incubation. Analysis of Cry1Ab protein in whole-plant corn using the ELISA technique revealed a decrease to 28.0% of the initial value within 2 h and to 2.6% within 48 h. The concentration of Cry1Ab protein of ensiled corn was only 10% that of whole-plant corn. Ensiled corn Cry1Ab protein decreased to 10% of initial values after 48 h of ruminal incubation. Using an immunoblotting technique, the full-size Cry1Ab protein was only detectable up to 8 h; thereafter, only fragments of approximately 17 and 34 kDa size were found. In conclusion, ruminal digestion decreased the presence of functional cry1Ab gene fragments. It is unlikely that full-size, functional Cry1Ab protein will be present after 8 h of incubation in the rumen. Therefore, results based on ELISA measurements should be interpreted carefully and verified by another detection method that discriminates between the full-size and fragmented Cry1Ab protein.
Wiedemann
S
S
Physiology Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, D-85354 Freising, Germany.
Lutz
B
B
Kurtz
H
H
Schwarz
F J
FJ
Albrecht
C
C
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D002417:Cattle / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D018744:DNA, Plant / Q000378:metabolism*; D004032:Diet / Q000662:veterinary; D004731:Endotoxins / Q000378:metabolism; D005260:Female; D006460:Hemolysin Proteins; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D012417:Rumen / Q000378:metabolism*; D013997:Time Factors; D003313:Zea mays / Q000378:metabolism*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018744:DNA, Plant; D004731:Endotoxins; D006460:Hemolysin Proteins; D010940:Plant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012273:Ribulose-Bisphosphate Carboxylase