Study On Glyphosate Resistance Gene AroAM1 Transformation Of Tobacco

Glyphosate,developed in 1970s,is one of the most successful herbicides in the world.It makes it possible to be applied far-ranging and one of the best-selling herbicide since it has the character of broad character,do no harm to environment and health of life form and can eradicate perennial plants which depend on roots for reproduction.Recently,this herbicide developed very fast,which opens up extensive application prospect for glyphosate-resistant transgenic plants From the beginning of cultivation of glyphosate-resistant transgenic soybean in 1996 in USA,the transgenic plant is increasing every year in the world.However,almost all of the glyphosate-resistant transgenic soybeans are Monsanto's patents.Therefore,developing transgenic plants of our own intellectual property rights and making their commercialized development realized will be the key point in the domestic study of herbicide-resistant crops.Gene aroA can encode EPSPS-one of the key enzymes during the action of glyphosate. The gene aroAM1 used in our study originated from the recombination of gene aroA of Salmonella typhimurium and Escherichia coli.This study takes agrobacterium tumefaciens as intermediate mediator and makes the injured tobacco leaf cell affected through agrobacterium-mediated transformation method.Then,we integrate plasmid DNA fragments into the genome of tobacco cell in order to get transformed endogenous objective gene aroAMl. In the end,we get the transgenic tobacco ones which are cultivated by the tissue cultivation technology.On the basis of obtaining transgenic agrobacterium tumefaciens,we cultivate tobacco leaf disc in the optimized medium which is contain 0.1μg/ml IAA,2μg/ml 6-BA,500μg/ml CB, 24μmol/L glyphosate,and harvest the regenerate tobacco plants.Then,we detect the three different levels of regeneration of tobacco plants:on molecular level,positive rate of PCR is 100%;on organic level,resistance of transgenic in vitro tobacco leaves against glyphosate increases by 12 times;on entire plant level,when smeared 5mmol/L glyphosate on the leaves, the non-trangenec tobacco is more sensitive to glyphosphate than transgenic ones.The results indicated that,on the basis of the optimized glyphosate-resistant medium,we can selectively cultivate the tobacco and obtain transgenic tobacco plants.In the future,due to the glyphosate with characteristeristics of safety and non-polluting,as well as the functional verifcation of the gene aroAMl transforming into the model plant tobacco,the gene can be in-depth studied of the gene in transgenic plants on location,expression of object protein EPSPS,and mechanism of glyphosate tolerance in transgenic plants.