Construction Of Brachypodium Distachyon CBF2 Gene Expression Vector And Resistance Analysis Of Transgenic Arabidopsis Thaliana

This study use Brachypodium distachyon CBF2 gene and Arabidopsis thaliana as the object, construct the expression vectors pK24-35S-CBF2 of CBF2 gene by using gateway technology, through agrobacterium mediated method, and transformed CBF2 gene into Arabidopsis thaliana. After screening the three generation seed of transgenic plants by tissue culture,positive homozygous plants obtained. Use them for the study of physiological and biochemical reaction under the dehydration、saline-alkali stress during the seed、seeding and plant stage.To discuss reactivity and the function of CBF2 gene.1. The plant expression vector pK24-35S-CBF2 of CBF2 gene was constructed by the gateway technology. Brachypodium distachyon CBF2 gene was transferred into Arabidopsis thaliana. through the method of Agrobacterium tumefaciens EHA105 mediated.13 transgenic Arabidopsis thaliana positive plants in T1 generation were obtained through kanamycin screening.7 transgenic Arabidopsis thaliana lines were obtained via the second and the third generation screening, and their germination rate and the conversion rate were over 80%.7 transgenic positive Arabidopsis thaliana lines were identified by PCR and the target bands were observed.2. The seeds and seedlings, whose germination rate and the conversion rate were more than 90%, were conducted drought experiment. The results showed that there were no significant differences of the drought resistance in the seed stage between the transgenic and the wild type plants. During the period of seedling, the growth of transgenic Arabidopsis thaliana root length was significantly higher than the wild type’s when mannitol concentration was 100、150、200、250、300mmol/L, and it indicated that the drought resistance was strengthened during the seedlings period of the transgenic Arabidopsis thaliana. But during the seeding stage treated with drought stress, the transgenic Arabidopsis thaliana plants and the wild type in phenotype showed some differences. MDA and electrical conductivity values were significantly lower than that in wild type plants, cell membrane was damaged lighter than the wild type Arabidopsis, and the POD enzyme activity is always higher than the wild type Arabidopsis after the treatment of 8-20d. These results showed that the CBF2 gene enhanced the ability of drought resistance of transgenic at the seedlings and seedling stage in part.3. Seeds and plants of T3 germination Arabidopsis thaliana were treated with salt. Results showed that the germination rate of transgenic tobacco lines was significantly higher than that of wild plants under the 150mmol/L of NaCl concentration. In seedling stage, the root growth of transgenic plants was significantly higher than wild one with increasing NaCl concentration and stress time; while for the 150 and 250 mmol/L of NaCl concentration, the longer processing time, the stronger of salt-tolerance of transgenic plants will have. The SOD and POD enzyme activity of transgenic plant were much higher than wild one, and the trend leveled off in the mid-and late processing. The content of MDA and relative conductivity of transgenic plants was lower than wild one. This indicated that the regulation of CBF2 transcription factor enhanced the ability of transgenic plants removing active oxygen radicals, and relieved the membrane lipid peroxidation of transgenic plants under the salt stress. Overall, the expression of CBF2 gene effectively enhanced the salt tolerance of transgenic plants in each growth stage.4. The transgenic seeds and plants of T3 generation were treated with NaHC03 stress. Results showed that the germination rate of transgenic tobacco was significantly higher than wild plants at the 7.5 and 10 mmol/L of NaHCO3 concentration. In the seeding stage, the root growth of transgenic Arabidopsis thaliana was significantly higher than wild one under the NaHCO3 concentration of 2,5,7.5, and 10 mmol/L. With the increase of concentration of NaHCO3, the phenotypes difference between transgenic and wild plants became significant, and the transgenic plants have higher survival rate. After transgenic seedling was treated by NaHCO3 with different concentration their SOD and POD activity were significantly higher than wild one, and the content of MDA and relative conductivity was lower than wild one. This Indicated the stability of plant cell membrane. In short, the transgenic plant has stronger resistance to NaHCO3 than the wild one in each growth stage.