Project Number: 5335-21000-031-00
Project Type: Appropriated

Start Date: Mar 27, 2006
End Date: Mar 26, 2011

Objective:
Objective 1: Develop and evaluate transformation vectors that use site-specific recombination systems for more efficient and precise integration of DNA into the host genome. Test the site-specific integration and repeated stacking of DNA into designated chromosomal sites using selected reporter genes and oxidative stress tolerance genes. Objective 2: Develop and evaluate the use of site-specfic recombination to break the linkage drag associated with the introgression of transgenes from laboratory lines to field cultivars. Test the reciprocal exchange of transgenic DNA flanked by recombination sites.

Approach:
For objective 1, vectors will be constructed by recombinant DNA methods using as much as possible publicly available genetic components. Vectors will be designed for use in direct DNA transformation. Model dicot and monocot plant systems will be transformed with the initial empty vector harboring a reporter gene. The initial transformants will be screened for single copy insertion and acceptable expression level of the reporter construct. Several of the initial transformants that meet the above criteria will be propagate as "target lines". Progeny of these target lines will be used to test the site-specific integration of additional DNA into the same locus, mediated by the transient co-introduction of the appropriate recombinase gene. Site-specific integrants will be subjected to further introduction of a second recombinase gene to remove unneeded DNA. Characterization of the structure and expression of the introduced DNA will be performed by standard molecular analysis. For objective 2, a construct will be made by recombinant DNA methods with sets of recombination sites designed for interchromosomal recombination and harboring a first marker gene. This construct will be transformed into line #1. The transgenic locus of the line #1 will be introduced into another genetic background such that the flanking DNA of the new line #2 differs from that of line #1. The transgenic locus of line #1 will be further modified through site-specific insertion of a second marker gene to form line #3. Line #3, with first and second marker genes, will be crossed to line #2 containing the first marker gene. The efficiency of exchange by site-specific recombination between the two transgenic loci will be examined in progeny, as determined by linkage to polymorphic flanking DNA. Formerly 5335-21000-016-00D (4/06). BSL-1; 2/19/08.