Reduction of transmission of transgenes in plants
A technology of transgenic plants and plants, applied in plant products, genetic engineering, botany equipment and methods, etc., can solve problems such as failure
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Embodiment 1
[0055] This example illustrates the isolation and mutagenesis of the atDMCl promoter. Turning now to Figure 3, it shows the known restriction map of the Arabidopsis Ecotype Lansburg, and a cloning scheme for the isolation of the atDMC1 promoter region. The atDMCI promoter of the present invention can be generated using standard molecular genetic cloning methods and polymerase chain reaction (PCR) known in the art. Genomic DNA was isolated from Arabidopsis Ecotype Lansburg and PCR amplified using primers DMC1 (5'-gttaacaccgtttatatgagacaaaatcagctatg-3'; SEQ ID NO: 1) and DMC4 (5'-catceccacttgcgaattcactacc-3; SEQ ID NO: 2). The resulting product was then digested with restriction enzymes HindIII and EcoRI to generate a HindIII / EcoRI DNA fragment. Genomic DNA was then amplified by PCR with primers DMC3 (5'-ggtagtgaattcgcaagtggggatg-3'; SEQ ID NO: 3) and DMC2 (5'-tgctctagactcgctctaagactctctaagctaggaagagtgagag-3'; SEQ ID NO: 4), and the resulting product was amplified by EcoRI Dig...
Embodiment 2
[0057]This example illustrates the construction of a NOS blocking sequence between two FRT sites. Turning now to Figure 2, it shows a schematic diagram of the cloning method that can be used to obtain blocking sequences between FRT sites. The FRT site of this example contains a minimal FLP recognition sequence and additional flanking repeats to ensure efficient recombination. Lower case letters in the FRT sequence represent restriction enzyme specific sequences, while upper case letters represent FRT nucleotides. Plasmid pMUCBS (this is pMUC9 with pBS+(KS) polylinker; Jones, J. et al., 1985) was digested with BamHI and PstI restriction enzymes, and ligated to FRT-3 / FRT-4 shown in SEQ ID NO:5 sequence. The FRT-1, FRT-2, FRT-3 and FRT-4 sequences shown in the figure are synthetic oligonucleotides, which were synthesized using standard methods well known in the art. The resulting DNA was then digested with XhoI, filled in to remove overhangs, digested with PstI and ligated to ...
Embodiment 3
[0059] This example illustrates the construction of a DNA sequence containing the transiently expressed atDMCI promoter and Cre gene separated by a blocking sequence between the FRT site-specific recombination sequences. Turning now to Figure 5, it shows a schematic diagram of the cloning strategy used to construct the DNA sequence containing the Cre gene under the control of the atDMCI promoter. The plasmid generated in Example 1 was digested with HindIII / XbaI. Plasmid pED23 (Dale et al., 1990), which contains the 35S promoter operably linked to a Cre recombinase gene which is further operably linked at its 3' end to a NOS terminator, was also digested with HindIIII / XbaI. sequence. The two digests were then ligated, digested with XbaI, and filled in to remove overhangs. The block sequence between the FRT sites of Example 2 was also digested with SpeI, filled in to remove overhangs, and then digested with KpnI. The two resulting DNA fragments were ligated to generate the fo...
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