Construction Of A Marker-free Practical Binary Vector By Using The Cre/loxP System

With the development of plant biotechnology, genetic transformation of crop plants is becoming increasingly routine and shows its potential application value in crop breeding. As we all know, antibiotic resistance genes are commonly used as selectable markers for the selection of transformants from a large number of untransformed cells.However, once transgenie plants are regenerated, antibiotic resistance genes serve no useful purpose but they continue to express their gene products . Consequently, more and more environmental and consumer concerns regarding the safety of the transgenic products containing selectable marker genes(SMG)in transgenic plants, at least there were be three major problems: (1)the marker gene may have negative effects on proliferation and differentiation of plant cells; (2)there is uncertainty regarding the environmental impact of many selectable marker genes: (3)it is difficult to perform multi-desirable genes transformations using the same selectable marker. In order to solve these problems, many approaches have been developed to obtain marker-free transgenic plants (MFTPs), which has shown a tendency to eliminate any marker gene DNA sequences in the final product.DNA recombination mediated by Cre remmbinase has become an important tool to generate marker-free transgenic plants (MFTPs) because it is time-specific, tissue-specific and site-specific. The Cre / loxP system of bacterio-phage P1 is a site-specific recombination system that consists of two components: the recombinase (Cre)and its recognition sites (loxP). Cre mediates recombination events and causes the excision of the DNA segment between two directly adjacent loxP sites.A novel practical binary vector to get marker-free transgenic plant was constructed by modifing the plant expression vector pRAL-MCS. The heat shock-inducible Cre / loxP DNA recombination system was adopted in this system by using the promoter of Arabidopsis thaliana heat shock protein 18.2. All selectable marker genes (GFP and BAR) and cre enzyme gene located between two identical orientation loxP sites could be excised from the transgenic genome by the Cre expression. The selectable marker genes: GFP and BAR were under the control of two constitutive promoters: Adhl promoter and CaMV 35S promoter respectively.The above newly created plant expression vectors can be used to develop selectable marker-removable transgenic plants for a variety of purposes.