Browsing by Subject "MON810"

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Biochemical investigations on genetically modified oil crops
(2009) Mekawi, Enas; Schwack, Wolfgang
The main purpose of this study was to develop a method of purification and characterization of Cry1Ab isolated from MON810 genetically modified maize. The second object was to study the effect of the genetic modification of MON810 and high-oleic sunflower on the oil composition. Therefore, the following investigations were performed: (1) Quantification of Cry1Ab toxin in different corn plant parts. (2) Development of a suitable method for purification of Cry1Ab from MON810. (3) Establishment of characterization method for Cry1Ab by mass spectrometry with regard to high peptide sequence coverage. (4) Evaluation of the effect of genetic modification on the oil composition compared with the conventional traits. The following results were obtained: Screening of Cry1Ab by ELISA is the most predominant technique for determination of Cry toxin content in plants. The determination of the toxin concentration resulted in highest levels for leaves (26.8 µg/g dry matter), while it was 1.5, and 1.0 µg/g for stalks and grains respectively. In our study, toxin content in leaves was about six times higher than in a previous study. There are no data available for the dry weight content of Bt toxins in stalk and grain, which could be compared to the obtained results. Although MON810 maize is one of the major genetically modified crops, informations on the character of the Cry1Ab purified from the MON810 maize is still limited, although such data are important for safety assessment studies. To my best knowledge, this study is the first investigation charactizing Cry1Ab toxin isolated from MON810 maize. The results of the present investigation indicated that the separation of the Cry1Ab protein from MON810 leaf extracts by HPLC techniques was not efficient. MALDI-TOF analyses showed that the major component separated with Cry1Ab was β-D glucosidase, which may be due to resembled isoelectric points. However, immuno-affinity purification using self-prepared affinity columns was very efficient to isolate pure Cry1Ab from MON810. The characterization of purified Cry1Ab was successfully done by SDS-PAGE, Western blot analysis and MS techniques. MALDI-TOF MS analyses were useful for component screening of Cry1Ab. Results showed that Cry1Ab is subjected to truncation by plant proteases into a core toxin with approximately 69 kDa. LC(ESI)-MS/MS gave a higher sequencing coverage of Cry1Ab (73 % of peptide sequence) compared to MALDI-TOF analysis (41% of peptide sequence). Further studies revealed that Cry1Ab had no detectable potential carbohydrates which might be covalently linked to the protein. The capillary electrophoresis technique was used for determination of the Cry1Ab purified from MON810 maize and proved to be a suitable method for determination of the Cry1Ab, but it was not successful for the detection of very low quantities (less than 0.03 mg/ml). Peptide mapping is one of the most powerful tools for protein identification and characterization. The use of HPTLC with the relatively new plates (ProteoChrom) was identified as a convenient tool for peptide mapping as compared to capillary electrophoresis, especially if put into consideration that HPTLC is less costly than capillary electrophoresis. The HPTLC method was able to resolve 13 peptides, while capillary electrophoresis resolved 19 peptides, obtained from the digested Cry1Ab toxin. Concerning lipid analyses, fatty acids and sterols were determined by gas chromatography, tocopherols by HPLC. For the determination of phospholipids, an HPTLC method was developed, resulting in lower detection limits than reported in previous studies. The present study proved that the genetic modification did not significantly affect the contents of fatty acids, sterols, tocopherols and phospholipids in transgenic maize oil. Apart from the increased amount of oleic acid in high-oleic sunflower oil, the genetic modification in sunflower did not produce unexpected effects on the oil composition. Therefore, with regard to the oil composition, both oils from genetically modified plants will be as safe as conventional oil types.