Studies On Expression Of Superoxide Dismutase Gene (Mnsod) And Transcription Factor Gene (dreb) In Maize

Maize originates from the tropical zone of high temperature and humidity in Central and Southern America, so that it has poor tolerance to drought and low temperature stress. With the aggravation of ecological conditions, drought has become the major limitative factor to maize production in many countries and regions. Under stress conditions such as drought and low temperature, oxidative metabolism is hindered in plant, because of inhibition of endogenous system of active oxygen extinguishing, such as superoxide dismutase (SOD) isozymes. A great deal of activate oxygen is accumulated and damages protein, membrane lipid, DNA and other components. So that the physiological and biochemical metabolism is disturbed and the growth and development of plant is seriously impaired. It is indicated that the activity of superoxide dismutase is positive correlation with tolerance to antioxydation and can be used as a criteria of stress tolerance identification in maize.In order to evaluate the effect of over-expressed Mn-superoxide dismutase gene on antioxydation in maize, expression vector with Mnsod gene from wheat was constructed for monocotyledonous plant and used to transform calli of elite maize inbred line by microprojectile bombardment. Plants were differentiated and regenerated from the positive calli screened with gradient concentration of hygromycin. PCR, Southern hybridization and SOD activity assay displayed that exogenous gene was integrated in five maize genomes, exogenous protein was over-expressed in partial plants and the activity of MnSOD of the transformed plants is more than 3 times of the untransformed control. The highest activity is as high as 9.4 times of the control.Antioxydation ability of the transgenic plants was detected with the method of electrolyte leakage ratio after oxidative damage treatment with methyl viologen. The result showed that the electric conductivity of all the transgenic plants was lower than the control, except one of them. Plant line number 2 had greatly significant difference (P<1%) with the control. It was indicated that antioxydation ability of the transgenic plants with over-expression of Mnsod gene was improved significantly under stress conditions and could be used to breed varieties with drought, salt and low temperature tolerance.In recent years, transcription factors of stress resistance have become a hot spotof plant molecular biology research. This is useful to understand stress transduction in plant cells and expression regulation of responsive genes. Cis-acting element DRE, with core sequence of CCGAC, is found in many promoters of stress resistant genes. This sequence is recognized and bound specifically by transcription factor DREB, to promote the expression of down-stream genes. Therefore, over-expression of transcription factor DREB can induce expression of the genes with cis-acting element DRE and improve extensive resistances of crops.In this study, a stress-inducible promoter mwcs!20 was cloned from wheat by specific PCR. 97.1% homology of this promoter was found with promoter wcs!20 and no change was found with conserved domain of stress regulation, although base mutation was found at two sites. The transient expression experiment with gus gene showed that mwcsllO promoter was induced by high salt and low temperature and promoted expression of structural genes in both monocotyledonous and dicotyledonous plants. It is hopeful to be used in gene engineering of stress resistance in crops. The sequence of this promoter was registered at GENBANK with accession number AY493570.Transcription factor gene of stress resistance dreb was promoted by inducible expression promoter mwcs!20 and constitutive expression promoter ubiquitin respectively. Inducible and constitutive expression vectors for monocotyledonous plants were constructed and used to transfer into maize calli. Plants were differentiated and regenerated from the positive calli screened with gradient concentration of hygromycin. 4 transformed plants with inducible expression vector and 2 with con...