Construction Of Superpromoter And Expression Vector

Although octopine synthase (Ocs) and mannopine synthase (Mas) genes initally resident on Ti plasmid of Agrobacterium tumefaciens, they possess all of the sequence elements required for transcription in plants. For many years, ocs and mas gene promoters have been used by plant molecular biologists to direct the expression of linked genes in transgenic plants. In this study, we combine ocs activator elements with mas promoter, then fuse the chimeric promoters on GUS gene to construct plant expression vectors. We also demonstrate by comprison to the 35S promoter. Leaf disks from tobacco are infected with cultures of A. tumefaciens transconjugants harboring the different plasmids, and transgenic plants are generated by the leaf disk procedure, plant extracts are analyzed by the fluorescent method for detecting GUS activity. Conclusions are drawn from our experiments and shown below:1. Of the combinations we tested, the construction containing four copies of ocs activator resulted in the highest level of expression, which strongly elevated the expression of GUS activity in leaf (23-fold) and root (47-fold) compared with 35S promoter.2. Odd or even copies of ocs activator follow the rule that addition of multiple ocs activators upon mas promoter increases promoter strength. However, even copies of ocs activator enhance promoter strength higher than odd copies of ocs activator. In order to explain results above we propose a model that multiple copies of activator interact with promoter via contacts between DNA-bound transcription factors.3. There is no difference in range of GUS activities using constructions in which the activator elements are cloned in opposite orientation.4. Individual transgenic plants containing the same construction exhibited a wide range of GUS activity because of position effect.5. Constructions containing even copies of ocs activator show apparentorgan- specific promoter activity. However, chimeric promoters harboring odd copies of ocs activator is constitutively active and not strongly organ- specific.6. All of chemeric promoters are able to direct a basipetal pattern ofexpression.7. The expression of all the chemeric promoters is enhanced by wound. However, the degree of wound induction is different.