| The transgenic technology, with the development of plant gene-engineering, has been a very important approach to modify the plant genetic characters. However, it is also a potential hazard to the environment and human health when making use of bio antibiotic or herbicide resistant genes as the selectable marker genes during transformation, which limits the prospects of the application for commercial production. Using the no-doubtful safe selectable marker genes is one of the practicable methods to solve the problem. For the purpose of genetic improvement of plants through Agrobacterium-mediated transformation or bombardment using particle inflow gun, different plant expression vectors carrying manA as the safe selectable marker gene with different promoters and target foreign genes have been constructed in this study. The main results are as follow:1. The manA gene coding for a phosphomannose isomerase (PMI) with 42 KD was cloned from the Escherichia coli isolate DH5αand sequenced. It was then integrated into a prokaryotic expression vector pGEX-6P-1. Upon the induction with IPTG, abundant protein with the expected molecular weight of 68 KD was expressed and affinity-purified. The purified protein was cleaved with Prescission protease and two distinct proteins, a 26 KD of GST and a 42 KD of PMI, were identified on SDS-PAGE, confirming the correct size of the fusion protein.2. Phenol red assay indicated the growth situation of the Escherichia coli BL21(DE3) when they made use of mannose as the exclusive source of carbon. Inclusion of various mannose concentrations in media revealed that at the concentrations of 30% and 40% of mannose sugar the cell growth of BL2 (DE3) was significantly inhibited. And at the concentration of 30%, functional PMI activity in BL2 (DE3) was detected with the color changes of phenol red chemicals. These results indicated that the cloned gene manA can be functionally expressed in batacterium as the enzyme PMI.3. The plant expression vectors were constructed that carry the manA as a safe selectable marker gene and were fused to different promoters respectively. The vectors can be used both in Agrobacterium-mediated transformation and bombardment of particle inflow gun. 14 different combinations of Fusarium-specific antibodies and antifungal peptides were integrated into the vectors for Agrobacterium-mediated transformationn under the control of Maize Ubi-1 promoter. In total 13 cloning vectors and 17 plant expression vectors carrying different target genes were constructed in this study.4. The proper conditions such as mannose concentrations used for selection of positive transgenic Arabidopsis plants with PMI/mannose system have been studied. According to the growth and development of Arabidopsis thaliana, very few Arabidopsis plant can develop normally at the sugar concentration of 4.4g/L mannose plus 25.6g/L sucrose after germination, and therefore this sugar concentration was chosen as the proper pressure for the selection of transgenic Arabidopsis plants.5. One constructed vector carrying the manA as a selectable marker gene under the control of CaMV35SS promoter was used in Agrobacterium-mediated transformation of Arabidopsis via floral dipping. 10 transgenic Arabidopsis plants, which were obtained under the selection condition of 4.4g/L mannose plus 25.6g/L sucrose, were random sampled for Chlorophenol red assay and 7 plants were positive. PCR analysis verified that manA gene was integrated into the genome of the 7 plants. These results demonstrated that the plant expression vectors constructed in this study that contain manA as a safe selectable marker gene can indeed be used in plant transformation with the selection agent of mannose. |
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