Functional Evaluation Of Two Stress-Responsive Promoters In Maize

Maize is under stress of drought, low temperature, saline, and many other abiotic adverse conditions during its whole growth proves. Its growth and development, as well as yield, are affected seriously. Breeding new resistant varieties by transgenic technology is a key to solve these problems. However, the resistant genes were promoted by constitutive promoters in many transgenic researches. Although the resistance to abiotic stresses was improved, the energy waste caused by overexpression of the resistant genes inhibited the physiological and metabolic activities of the transgenic plants under non stress conditions, resulting in dwarf plants and yield reduction. Because of the species specificity, the promotion activities of the exogenous inducible promoters are generally less than the endogenous promoters. At present, a few literatures are available about inducible promoters in maize. Cloning and function evaluation of maize endogenous inducible promoters are useful for understanding mechanism of stress response, and transgenic researches for stress resistance in maize.In the previous study, MGL3s and sbCBF6s promoters were cloned from maize, and evaluated by transient expression by transient expression to have multiple inducible activities in response to abiotitc stresses. The inducible activities of MGL3s are lower under non stress conditions than stress conditions such as drought and saline. The inducible activities of sbCBF6s do not response to drought, saline, ABA and some other abiotitc stresses, except low temperature. For further evaluation of inducible activities of these two promoters under abiotic stresses, as well as the effects of the cis-acting elements to the inducible activities, the analysis software of plant promoters PlantCARE was firstly used to identify TGACG, MBS, CGTCA, ARE, HSE and some other cis-acting elements relative to osmotic stress and temperature. Then, PCR primers were designed based on the result, and used to amplify the 5’-end deleted promoter sequences without different members of the cis-acting elements for construction of expression vectors of GUS gene. These vectors were used to transform Arabidopsis thaliana or tobacco by the mediation of Agrobacterium. The T1 plants were treated with simulative drought (20% polyethylene glycol-6000), high osmotic (0.4 mol/L NaCl) and blank control, and induced for the expression of the GUS gene under the control of the MGL3s promoter. The result of GUS histochemical staining showed that the GUS gene under the control of the MGL3s promoter with the five different lengths of 5’-end deletion was weakly expressed in the vein or stem of the transformed Arabidopsis. The MGL3s and sbCBF6s promoters with different lengths of the 5’-end deletion promoted the expression of the GUS gene in the positive calli isolated from the transformed tobacco leaf discs on the resistant medium. Quantitative GUS of MGL3s 5’-terminal deletions indicated that the promoter activity is not detected. The shortest length that the MGL3s and sbCBF6s promoters maintained their promotion activities were 325 bp (from-175 bp to+150 bp) and 185 bp (from-77 bp to-108 bp), respectively. The above results indicate that MGL3s promoter is monocotyledon-specific. It weakly drives gene expression in dicotyledonous plant of Arabidopsis thaliana. The shortest length that the MGL3s promoter maintains its promotion activity is the 325 bp upstream from the+150 bp. The sbCBF6s promoter drives gene expression both in monocotyledonous and dicotyledonous paints. The shortest length that the sbCBF6s promoter maintains its promotion activity is the 185 bp upstream from the+108 bp.