| Concerns have been raised that the presence of selectable marker genes such as antibiotic or herbieide resistant genes might be unpredictable hazard to the ecosystem as well as to human health. Marker-free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the seletable marker genes conferring antibiotic or herbicide resistance and so on.In this study, Marker-free vector pX6-NPI and pX6-GFP-NPI containing interest gene were constructed by inserting NPI into pX6-GFP, adopting the method of site specific recombination when the marker-free was removed.The result of PCR and enzyme cut indicated the two vector were completely construced.Then, the transgenic tobacco and NL-895 were studied and the main results are as follows:1. Marker-free vector pX6-GFP can insert interest genes and can be applied to transgenic jobs.2. The choose of enzyme cut site is diffcult, so middle plasmids must be used.3. In this experiment. the Agrobaclerium is EHA105, which its activity is quite strong. In the transgenic process, we established four kind of Cef gradient (50mg/L , 100mg/L, 150mg/L, 200mg/L),and the tobacco were carried on the Cef sensitivity test. The result indicted that Cef 50mg/L could not restrain Agrdbacterium growth, but Cef 150mg/L could restrain Agrobacterium growth very well.4. In the experiment, which Used the constructed Marker-free vector in tobacco, the differentiation frequency are all 100%. Then we obtained 12 transgenic plants of pX6-NPI and 10 tarnsgenic plants of pX6-GFP-NPI. PCR analsis result of transgenic tobacco proved that the NPI gene had existed in the tobacco.5. In transgenic experiment of NL-895, the result indicted the differentiation frequency of the poplars were all lower than the usual transgenic experiment of poplars.The cause maybe that the two vector is very big ,which are all 15000bp. |
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