A BglII+EcoRI digest produces two fragments from the insert. The approx. 330 bp BglII/EcoRI fragment contains 5' non-coding region and sequences encoding a chloroplast transit peptide and the 5' end of EPSPS. The 1.62 kb EcoRI fragment encodes the 3' end of EPSPS and contains the poly(A) tail. pMON546 contains a nopaline synthase/neomycin phosphotransferase II fusion gene that confers kanR only in transformed plant tissue. Both pMON546 and pGV3111SE can be isolated from the A. tumefaciens culture by co-culturing with E. coli containing a helper mobilization plasmid like pRK2013. E. coli with pMON546 are spcR strR kanS, while E. coli with pGV311SE are spcS strS kanR. pGV3111SE is a crippled Ti plasmid (the region containing tms tmr tml and OCS has been deleted) that contains vir genes which will integrate T-DNA sequences into a host chromosome. When Petunia cells are transformed with A. tumifaciens containing pMON546 and pGV3111SE, the CaMV/EPSPS gene permits growth in the presence of 0.5mM glyphosate. Phenotype verified: kanR, spcR, strR, cmlR. |
