Construction Of FLP/frt Site-Specific Recombination System And Its Application In Transgenic Tobacco

Transgene technology offers significant advantages for modern agriculture. Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance. Due mainly to consumer concerns, a suite of strategies have been developed to either avoid or get rid of 'problematic' selectable marker genes before transgenic plants are introduced into the field. One way to get the marker-free transgenic plants is to excise the selectable marker gene out of the integrated transgene after successful selection. In the FLP/frt site-specific system of the 2μm plasmid of Saccharomyces cerevisiae, the FLP enzyme is a 48kDa protein that covalently binds as four monomeric units to two frt sites and catalyzes the cleavage and ligation of these sites. FLP-mediated DNA excision or inversion occurs depending on the orientation of the frt sites and when they are in the same molecule; the FLP recombinase catalyzes integration of two DNA molecules when the frt sites are in two different molecules. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker gene from the nuclear genome after selection.In this study, the plant FLP/frt site-specific recombination system was constructed that included the FLP expression vector pCAMBIA1300-35S-FLP-hpt, the frt containing vectors pCAMBIA1300-AtMHX1-f/t/-als-frt and pCAMBIA1300-betA-frt-als-frt. In co-transgenic plants, the marker gene als flanked by two identical orientation frt sites could be excised by the constitutive expression of FLP recombinase under the control of CaMV35S promoter. In order to improve the efficiency of this system, the plant optimal translational modification sequence AACA was immediately adjacent upstream of the ATG initiation codon of the FLP gene , and one full-length frt site and one modified frtsite composed of only core recombination sequence were induced to the flank of als gene.The function of this system was tested in the transgenic tobacco plants by re-transformation or co-transformation method.1. The FLP recombinase gene was introduced into transgenic tobacco plants (AtNHXl/betA -frt-als-frt) by re-transformation. Excision of the als gene was found in 17% re-transgenic tobacco plants.2. Tobacco leaf discs co-cultivated with two different Agrobacterium LBA4404 that contained the FLP expression vector and the fit containing vector respectively. The highest frequency of co-transformants was 21% when the ratio of LBA4404 (jpCAMB\Al300-AtNHX\/betA-frt-als-frt) : LBA4404 (pCAMBIA1300-35S-FLP-/?p0 was 3:1, and 5% transgenic plants showed elimination of maker gene als.3. Transgenic tobaccoes carrying the marker gene flanked by two identical orientation frt sites and interest gene-AtNHX\/betA were re-transformed with the FLP expression vector. The frequency of re-transformation was 1.6%.The constitutive expression of FLP recombinase results in unwanted changes to plant genome. Based on several advantages of the heat shock promoter such as easy to manipulate, sensitive to heat shock treatment, high inductive activity, little effect on development of transgenic plants, it can be used to control the expression of FLP gene in transgenic plants. In this study, two soybean Gmhspl7.5E promoters were cloned. The sequence analysis of these two promoters showed that they have both basic elements of plant promoter and heat shock elements. Expression of P-D-glucuronidase under the control of these two promoters was investigated in transgenic tobacco plants after heat shock treatment. Results of histochemical stain assay and quantitative fluorometric assay indicated that Gus activity after heat shock treatment could reach 8 time and 4 time higher than that before heat shock treatment in line 63 and line 70 respectively.