Development Of Detection Techniques And Assessment Of Environmental Risks Of Transgenic Rapeseeds (Brassica.napus)

Ms8Rf3 is a herbicide-tolerant rapeseed hybrid developed by Byer crop-science company with transgenic sterile line Ms8 (Barnase and bar genes introduced) and transgenic restorer line Rf3 (Barnase and bar genes introduced) as parents. This hybrid has been commercialized and it occupied over 45% of the rapeseed planting area in Canada. During recent years, seeds of this hybrid have been imported by trade into China at a large quantity. However, the event-specific detection method of Ms8Rf3 has not been established yet, and the ecological risks of this hybrid have not been evaluated in our ecological environment, which becomes the bottleneck of the bio-safety management of transgenic rapeseed in our country.This study is designated 1) to develop event-specific PCR method and ELISA method for Ms8Rf3 detection; 2) to assess the possibility of foreign genes transferring from the transgenic rapeseed to related native species; 3) to assess the genetic stability of Ms8Rf3 in China's environment, so as to provide a technical support and scientific basis for our government to implement GMO bio-safety management.Firstly, an event specific method for Ms8Rf3 detection was finally established through the study. The junction sequences of the foreign genes were successfully discovered, the event specific primers were designed and a high efficient PCR detection method was built up. Experiments demonstrated that samples containing Ms8, Rf3 or Ms8Rf3 can be efficiently found and distinguished. This method not only provides a technical support for GMO management but also filled the gap of this field.Secondly, the prokaryotic expression of bar gene was made and the purified PAT protein was obtained. This protein can be used for replacement of plant foreign protein PAT in experiment of food bio-safety assessment and also used for antibody preparation in ELISA detection, which are needed for biosafey assessment and GMO management. Bar gene of full length was isolated through PCR amplification from pCAMBIA1300-bar plasmid, which was identified by restriction enzyme cleavage. Recombinant plasmid pET28 bar was constructed by inserting bar gene into pET28a plasmid with BamHI and HindIII cleavage.After transformation of this recombinant plasmid to E.coli BL21(DE3),high efficient expression...