Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

摘要

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.