| Fruit ripening is associated with a number of physiological and biochemical changes including degradation of chlorophyll, carotenoid biosynthesis, synthesis of flavor compounds, conversion of starch to sugars, cell wall solublisation and fruit softening. These changes are brought about by the expression of specific genes, and promoters play a key role in transcription regulation of specific gene expression, wmll is a fruit-specific gene isolated from watermelon. It is an isoform of the gene encoding large subunit of ADP-glucose pyrophosphorylase (AGPase). The 5' promoter region of wmll, which covers 1864bp, has previously been isolated through the method of Uneven PCR. A transient expression assay has shown that a region with 1573bp is sufficient to drive fruit-specific expression of GUS gene. In this study, deletion analysis was conducted for the 1573bp region, and the main regulatory region in driving fruit-specific expression was targeted. In addition, plant expression vectors of Brazzein gene driven by fruit-specific promoters were constructed. The results obtained were summarized as follows:1) In accordance with the restriction map of the cloned 1573bp promoter region, three deletions were isolated using restriction enzymes. All these three deletions, which covers 1197bp, 896bp, 795bp, respectively, were subcloned into pBluescript SK (-) vector. These three DNA fragments were cut from the pBluescript SK ( - ) vector using Hind and BamHI, and inserted into plant transient expression vector pBI426, in front of the coding region of GUS to substitute for the constitutive CaMV 35S promoter.2) Fragments of 1573bp, 1197bp, 896bp and 795bp were introduced in front of the GUS-coding region to substitute for the constitutive CaMV 35S promoter in binary vector pBI121. And four vectors adapting for Agrobacterhtm-medialedtransformation were constructed.3) Tomato cotyledon explants were co-cultured with the tiimefacine strain GV3101 carrying four modified binary vectors, and regenerated plants were obtained. GUS-coding regions driven by these promoter fragments were verified to have incorporated into tomato genome through PCR specific amplification reaction and Southern-blot hybridization. Stems, leaves, root and fruits at different development stages from transgenic plants were used for GUS histochemical analysis, and GUS activity was determined. The results indicated that fragments of 1573bp, 1197bp and 896bp were able to drive GUS expression in a fruit-specific manner, with varying expression levels during fruit development. GUS expression in fruits of 30 and 45 days after pollination was strong, but in fruits of 15 and 60 days after pollination, the expression level was lower. The fragment of 795bp was not able to drive GUS expression in any of the organs analyzed. It could be concluded that the region between 857bp and 957bp contained the elements necessary for directing fruit-specific expression.4) Transient expression vector of GUS driven by four promoter fragments were delivered into the watermelon leaves, stems, flowers and fruits at different development stages (5, 10, 20 days after pollination) via particle bombardment. Histochemical assay showed that the 1573bp fragment could drive GUS expression in flowers, 5 and 20 days' fruits, but not in leaves, stems and 10 days' fruits. Fragments of 1197bp and 896bp could drive GUS expression only in 20 days' fruits. 795bp fragment was not able to drive GUS expression in any of the tissues bombarded. It could be concluded that there may be some cis-acting elements located in the region from 180bp to 556bp, which was involved with gene expression in the early stages of fruit development. Deletion of the region from 857 to 957bp led to loss of GUS expression, thus this region included the necessary information needed for gene expression as well as regulatory elements for fruit-specific transcription. In addition, with the help of syringe, transient expression of GUS gen...
|
PDF Full Text Request