Characterization And Transformation Into Tobacco Of Tomato DR8 Promoter

DR8 gene is one of the developmentally regulated (DR) clones, which are isolated following differential screening of gene expression during tomato fruit development. The 1031 bp promoter region of DR8 gene was amplified by PCR technique from tomato genomic DNA. It has been demonstrated that DR8 promoter was indeed induced by auxin, however, there isn't known auxin-response elements (AuxREs) in this promoter, so there is likely a new AuxRE. Sequence analysis showed that this cloned fragment has a lot of element, they may be some of the important regulation sites during the gene expression. But now we have not understood how this element work regularly. It's very important to research the regulation mechanism of the DR8 promoter.In this study, 4 plant expression vectors containing fragments of DR8 promoter were successfully constructed. Accompanied with the pBI121-GUS, these 5 vectors were transferred into tobaco. Differential expression of DR8 in transgenic tobacco organ were detected by analysis of GUS dyeing and GUS activity. Potential auxin-response elements has been analysised by treating with 2,4-D. At last, different treating including wounding, high temperature and ethylene were used to understand the changing of the DR8 promoter ability. The main results were as follows:①Here 4 plant expression vectors containing fragments of DR8 promoter based on pBI121-GUS were successfully constructed. The length were 1031 bp, 431 bp, 236 bp and 98 bp respectively. Their name were pDR8-1031,pDR8-431,pDR8-236 and pDR8-98 respectively.②Recombitant vectors were transferred into tobaco following the procedure of leaf-dish mediated by Agrobacterium tumefaciens. Transgenic plants were selected by using antibiotic and then identified through genomic DNA PCR, and also detected buy GUS dyeing. Transgenic tobacco were obtained.③The result of GUS dyeing and GUS activity showed that GUS gene expressed in the leaf of transgenic tobacco, but expressed very low or none expressed in the transgenic tobacco stem and root organs. The ability of DR8 promoter to drive the GUS gene is lower than 35S promoter.④Treating with 0.3 mg/L 2,4-D, 3 promoter, except pDR8-98, including pDR8-1031, pDR8-431, and pDR8-236, all enhance the expression of GUS gene obviously. It reveals that potential auxin-response elements location between -236 bp and -98 bp fragment of the DR8 promoter.⑤Wounding and treated with 100μL/L ethylene did not change the ability of DR8 promoter to drive the GUS gene express. But at the condition of high temperature, GUS activity was lower markedly. HSE did not enhance the ability of DR8 promoter to drive the GUS gene express.