Study On The Establishmednt Of Transgenic Insect-Resistant Assessment And Molecular Indetifying System

In recent years, the culture of GMC(genetically modified crops) has developed on a large scale all over the world and the main Chinese GMC turned into industrialization is insect-resistant cotton, which has bought great benefit to the society, but the safety of the transgene arouse more and more attention. The system of GMC identification has been established in China and now we report a insect-resistant cotton identifying system according to the transgene safety application of China at the molecular level. This is a basis reaserch of insect-resistant trangenetic cotton industrialization and our research may benefit to the molecular identification of GMC in china. The results are as below:1.A modified method for extracting total DNA from cotton has been accquired. This method can eliminate the disturbance of polysaccharide and polyphenol which is very rich in cotton effectively and the total DNA obtained by this method showed to be more integral and pure. The ratio of OD260 and OD280 is about 1.8 and the yield is about 15ng DNA per milligram tissue.2.We have established a PCR system which can rapidly identify Chinses insect-resistant cotton and America insect-resistant cotton. The optimal amount of the template is 50ng and the suitable condition is touch-down PCR:preequilibrated the PCR system to the denaturation temperature 94℃ for 7min and then,94℃ denaturation for 45sec, 63℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles； 94℃ denaturation for 45sec, 61℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；94℃ denaturation for 45sec, 59℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles； 94℃ denaturation for 45sec, 57℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；94℃ denaturation for 45sec, 55℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；94℃ denaturation for 45sec, 52℃ annealing of primer for 45sec, 72℃primer extension for 1min, 2 cycles；Followed by:Final extension:72℃ for 7min3.the the number of GFM cry1A copy in insect-resistant cotton genome has been detection by the method of Southern blotting and we suggest the optimal amount of genomic DNA for Southern is 30μg. The southern blotting show Bt toxin gene in 8 insect-resistant trangenetic cotton harbing single gene lines and 和5 insect-resistant trangenetic cotton harbing double gene lines is 1 to 4 copies respectively.4.The detection of GFM cry1A gene and cpti show the gene flow has not accur between insect-resistant transgenic cotton and other plant in the regions of insect-resistant transgenic cotton. 5. we have identify the expression of GFM cry1A gene at the protein level by special test strip and the result show GFM cry1A gene express differently in 13 insect-resistant trangenetic cotton lines.Postgraduate: Sun Xin Majo: BotanySupervisor: Prof. Hu Baozhong Prof. Guo Sandui...