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Methods and means for producing efficient silencing construct using recombinational cloning

  • US 7,732,660 B2
  • Filed: 01/13/2005
  • Issued: 06/08/2010
  • Est. Priority Date: 01/26/2001
  • Status: Active Grant
First Claim
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1. A method for preparing a plant comprising a final chimeric DNA construct, said method comprising:

  • preparing a final chimeric DNA construct capable of expressing a dsRNA of interest in a plant cell;

    said final chimeric DNA construct of interest comprising a DNA of interest (12) comprising a nucleotide sequence encoding said dsRNA of interest according to a method comprising the steps ofcombining in vitro a vector, an insert DNA and at least one site specific recombinase protein,said vector comprising the following operably linked DNA fragments;

    an origin of replication allowing replication in a recipient cell (1), a selectable marker region (2) capable of being expressed in said recipient cell; and

    a first chimeric DNA construct comprising in sequence;

    a promoter or promoter region (3) capable of being recognized by RNA polymerases of a plant cell;

    a first recombination site (4), a second recombination site (5), a third recombination site (6) and a fourth recombination site (7);

    a 3′

    transcription terminating and polyadenylation region (8) functional in said plant cell;

    wherein said first recombination site (4) and said fourth recombination site (7) are capable of reacting with a same recombination site, and said second recombination site (5) and said third recombination site (6), are capable of reacting with a same recombination site;

    and wherein said first recombination site (4) and said second recombination site (5) do not recombine with each other or with a same recombination site;

    orsaid third recombination site (6) and said fourth recombination site (7) do not recombine with each other or with a same recombination site;

    said insert DNA comprising said DNA segment of interest (12) flanked by a fifth recombination site (13) which is capable of recombining with said first (4) or fourth (7) recombination site on said vector; and

    a sixth recombination site (14) which is capable of recombining with said second (5) or third (6) recombination site on said vector;

    said at least one site specific recombination protein being capable of recombining said first (4) or fourth (7) and said fifth (13) recombination site and said second (5) or third (6) and said sixth (14) recombination site;

    allowing recombination to occur so as to produce a reaction mixture comprising product DNA molecules, said product DNA molecule comprising in sequence;

    said promoter or promoter region (3) capable of being recognized by RNA polymerases of said plant cell;

    a recombination site (15) which is the recombination product of said first (4) and said fifth (13) recombination site;

    said DNA fragment of interest (12);

    a recombination site (16) which is the recombination product of said second (4) and said sixth (14) recombination site;

    a recombination site (17) which is the recombination product of said third (5) and said sixth (14) recombination site;

    said DNA fragment of interest in opposite orientation (12);

    a recombination site (18) which is the recombination product of said fourth (7) and said fifth (13) recombination site; and

    said 3′

    transcription terminating and polyadenylation region (8) functional in said plant cell;

    selecting said product DNA molecules comprising said final chimeric DNA construct; and

    introducing said final chimeric DNA construct into at least one cell of said plant thereby preparing a plant comprising said final chimeric DNA construct.

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