Studies On Tissue Specific Expression Of Bt Gene And Molecular Detection Method Of Genetically Modified Rice

Since the first genetically modified crop came into being in the word, the genetically modified crops as a cluster of highest economic return crops have been widely spread in an incomparable speed throughout the globe. Rice, as one of important grain crops, was genetically modified in 1990s. However, the commercial program of GM rice has been restricted by the potential food safety, the ecological risk, intellectual property rights and product tagging etc. Many breeders are trying to seek molecular ways efficient and safe enough to avoid the risks of GM crops, in order to advance the step of GM rice commercial program. In this study, some attempts were put in the practice. For instance, tissue specific expression technique was applied GM rice so that we could control or avoid the risks of food safety, and molecular assay technique was developed to provide sufficient support for intellectual property rights and product tagging etc. A lot of main results were followed:In order to ensure the insecticidal genes do not express in edible parts of transgenic rice, two available promoters were obtained from six tissue specific promoters which were screened from rice materials including root, stem, leaf, flower, seed etc, by the means of RT-PCR technique. One was named Tsp I , and the other was Tsp II. After analysis of sequence and regular element of the promoters, the operation was performed, the GFP gene which localized in the pYP-Tsp I -GFP vector, was replaced with Tsp II or 35S. As a result two expression vectors which respectively contained Tsp II or 35S promoter were achieved.Two pairs of specific primers were designed according to the published sequence of Bt gene. The full sequence was amplified from genomic DNA template of insect-resistant rice variety "Keming Dao" by PCR. Sequence analysis showed that no differences were found between the cloned and the published sequences. The cloned Bt sequences was constructed in an pYP-Tsp I -GFP vector. The vector was used to transform.Gene-gun mediated gene transform method was developed and applied successfully in transformation process of the above vectors. Some Resistant was picked out in the transformed material "Wanjing 97".A construct-specific, qualitative detection method for the genetically modified rice based on the genetically modified materials, including these transgenic rice lines with bt, Xa21, ipt, pepc or bar gene, was established. Polymerase chain reaction (PCR) technique was applied to detect the introduced DNA sequences involved promoter, terminator, structural gene (encoding the novel protein), and other selectable marker genes. Compared with the protein detection methods for genetically modified crops, this method is more event-specific, sensitive and accurate.In addition, a quantitative method was developed for the genetically modified rice by real-time PCR with the specific primer and probe to amplify the 35S and NOS gene. Different protocols for real time PCR with probe Taqman and fluorescein SYBR Green I, and two quantitative detection methods for GM rice were systematically compared. From these studies, the real time PCR detection protocol for 35S and NOS genes and the quantitative protocol based on standard material in GM rice were established.