Agrobacterium-mediated Transformation Of Wheat Genetic And Resistance Of Tobacco

Iron is the necessary element for biological processes, and iron overload can cause oxidative stress, toxic to the cells, so maintaining iron balance in cellular is very important. Because of dual function of storage iron and release iron, Ferritin is the key protein to regulation of iron homeostasis in vivo. Researchers had cloned ferritin gene from the peas, alfalfa, Arabidopsis, and many other plants. Its’transgenic research focuses on import exogenous ferritin gene into plants to improve their resistance and iron content. ZHAO Yong-liang, etc. had cloned wheat ferritin gene(TaFer) and got transgenic yeast, found that the eukaryotic expression product of TaFer can effectively clear the three kinds of reactive oxygen species. To depth understand TaFer’s function of transgenic application, this study construct tobacco expression vector pBI121, by agrobacterium infection method, import TaFer into tobacco, study their resistance.Design primers according to the sequence AY864925of Genebank, join restriction site and His tag. Extract total wheat RNA, amplify TaFer by RT-PCR, the fragment named Fn. Fn connect to pGM-T cloning vector, transformed then screened by blue and white screening, transformants by PCR, double-enzyme digestion and sequenced, named Fn-pGM-T. Use the DNAStar software analysis the sequencing results, Fn match the size and sequence of AY864925, restriction site and His tag inserted correctly. Fn-pGM-T is double digest, pBI121to step digestion. Connect the two and transformed, transformants by PCR and single digestion, named Fn-pBI121.Transformed Fn-pBI121into agrobacterium, PCR identification results show that Fn-pBI121successfully transferred to Agrobacterium. Infection tobacco leaf discs after active the agrobacterium, after screening cultured10days, began to grow green callus. One month later, cut buds placed to rooting medium, roots after2weeks. RT-PCR results showed that TaFer has been transferred to tobacco. GUS staining showed that transgenic seedling expression GUS. Extract crude protein to electrophoresis, can see the target band.T1generation seedlings to stress(drought, salt, Cu2+) treatment, determination a variety of physiological and biochemical indicators before the stress, stress after10days, plant height20days after stress. Data show that:After drought stress, POD activity, soluble sugar and proline content of transgenic tobacco are higher than the wild-type. After salt stress, soluble sugar content, POD and CAT activity of transgenic tobacco are higher than the wild type. After Cu2+stress, POD activity and proline content of transgenic tobacco are higher than the wild-type. These results indicate that transfer TaFer to tobacco can increase its anti-stress capacity of drought, salt and Cu2+.