Progress 09/15/01 to 09/30/04
Outputs
Site-specific recombination involves reciprocal exchange between specific DNA sites catalyzed by site-specific recombinases, which provides tools for the development of a new generation of molecular technologies for crop improvement. The major objectives for this project were to produce transgenic rice lines expressing FLP recombinase and demonstrate in vivo the efficacy of FLP-mediated site-specific DNA recombination in crop species. To test the catalytic efficacy of FLP recombinase when expressed in crop species, we first obtained two sets of transgenic parental rice lines using FLP-containing construct pSB35S-bar/Ubi-FLP and the recombination-reporter construct, pSB35S-hyg/Ubi-FRT-neo-FRT-gus, respectively. In the pSB35S-hyg/Ubi-FRT-neo-FRT-gus construct, the presence of a blocking fragment, neo, flanked by directly oriented FRT sites between the ubiquitin and the gusA coding region prevented gusA transcription. Excision of the blocking sequence by FLP will bring
together the 35S promoter and the downstream gusA reporter gene, giving rise to gusA expression. A total of 516 T0 transgenic rice lines transformed with the FLP-containing construct, and 247 with the recombination-reporter construct, were produced using Agrobacterium-mediated transformation. Transient assay of GUS expression by bombardment of FRT-containing transgenics with an FLP plasmid construct, or by bombardment of FLP-containing transgenics with an FRT plasmid construct, was performed to visualize FLP-mediated site-specific DNA recombination in transgenic rice. Out of 159 plants bombarded, 97 showed GUS expression and the density of blue spots varied among treatments. The negative control did not show any GUS expression. The results obtained through this experiment demonstrated that FLP recombinase expressed in transgenic rice plants effectively exercised the DNA fragment flanked by the FRT target sites, resulting in site-specific DNA recombination and consequently, the GUS
expression. To test, in planta, the efficacy of FLP recombination activity when expressed in rice plants, we conducted cross-pollination experiments between the FLP-expressing plants and FRT-containing plants to bring together the FLP recombinase and the FRT-containing recombination-reporter construct in the hybrid. Based on molecular data obtained and results from transient GUS expression, as well as availability of pollen grains, we have conducted 28 crosses using 9 independent FLP-expressing T0 plants and 18 FRT-containing transgenic T0 plants. Seeds were harvested and germinated in the presence of both PPT and hygromycin to select the hybrid progeny inheriting both FLP and reporter gene gusA. GUS activity was evaluated in various tissues of the hybrids at different developmental stages. While all the hybrid progeny exhibited GUS expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Molecular analysis further confirmed the FLP-mediated
site-specific DNA excision in the hybrid. This observation clearly demonstrated the efficient operation of FLP recombinase in catalyzing site-specific DNA recombination, indicating that the FLP/FRT system functions in crop species.
Impacts
The results demonstrate the feasibility of using FLP/FRT site-specific recombination system as an efficient tool for genome modification in cereal crop species. This will enhance, dramatically, our ability to genetically improve a wide variety of crop species important to agriculture in the United States.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Various recombination systems have been evaluated for genetic manipulation in transformed plant cells, including the Cre/lox system of bacteriophage P1 and the yeast FLP/FRT system. While Cre/lox is well characterized, FLP/FRT has received considerably less attention. There are a number of applications for basic studies as well as crop improvement applications that would require two recombination systems that could be independently regulated in one transgenic organism. The objective of this USDA grant project was to produce transgenic rice lines stably expressing the yeast FLP recombinase and evaluate in vivo efficacy of FLP-mediated site-specific DNA recombination. We hypothesized that transgenic rice plants containing a recombination-reporter construct, pUbi-FRT-hyg-FRT-gus, in which the rice ubiquitin promoter and the reporter gusA coding region is separated by the hygromycin marker gene flanked by directly oriented FRT sites will not show GUS activity. When
crossed with plants containing stably expressed FLP recombinase, FLP should excise the blocking fragment (hyg gene) thus bringing together the ubiquitin promoter and the downstream gusA gene, giving rise to GUS expression in the hybrid plant. Using Agrobacterium-mediated transformation, we obtained transgenic plants with the FLP-containing construct or the FRT-containing recombination-reporter construct. We first conducted transient assays of the FLP recombinase activity using leaves from the transgenic rice seedlings. Transient assays for GUS expression in transgenic rice leaves containing the FLP gene after bombardment with the FRT-containing test plasmid pUbi-FRT-hyg-FRT-gus exhibited GUS expression. Reciprocally, the leaves of transgenic rice plants transformed with the FRT-containing test plasmid, pUbi-FRT-hyg-FRT-gus, when bombarded with the FLP gene construct, also exhibited transient GUS expression. These results demonstrated the in vivo functionality of FLP/FRT system in
transgenic rice plants. After cross-pollination between the FLP-expressing plants and FRT-containing plants, hybrid seeds were harvested and planted to produce F1 plants. Leaves were then sampled from the hybrid seedlings and stained for GUS activity. While the majority of the hybrid progeny exhibited a uniform GUS expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Detailed molecular analyses on all the DNA recombination events are currently under way, however, our preliminary results confirmed, at the molecular level, the in vivo FLP-mediated site-specific DNA excision in rice. The experimental data we have obtained so far demonstrate the efficient operation of FLP recombinase in catalyzing excisional DNA recombination in rice, indicating that the FLP/FRT recombination system functions in rice without any deleterious effects on plant development. This not only confirms the feasibility of using FLP/FRT system for genome modification in cereal
crop species, but also provides transgenic rice lines expressing FLP recombinase as foundational stock material, thus facilitating the future application and development of the FLP/FRT system in rice genetic improvement.
Impacts
The results obtained so far for the current project demonstrate the feasibility of using FLP/FRT site-specific recombination system as an efficient tool for genome modification in cereal crop species. This will enhance, dramatically, our ability to genetically improve a wide variety of crop species important to agriculture in the United States.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
We have proposed to produce transgenic rice plants expressing FLP recombinase and to evaluate the in vivo efficacy of FLP-mediated DNA recombination. To date we have synthesized two pSB 11-based Agrobacterium binary vectors for transformation of rice with a chimeric FLP gene and a recombination-reporter gusA gene. Using these two constructs, we have produced separate transgenic rice lines (callus and whole plants) expressing FLP recombinase or containing the recombination-reporter gusA gene. We have also evaluated the efficacy of FLP-mediated DNA excision in vivo by measuring GUS activity in transgenic callus carrying the recombination-reporter gusA gene after bombarding the callus with the chimeric FLP gene construct using a gene gun. Results indicate functioning of the FLP construct to produce multiple blue spots on the callus. We are currently producing hybrid plants from the cross between the regenerated transgenic rice plants expressing recombinase FLP and plants
carrying the recombination-reporter gusA gene. We anticipate observing efficient functioning of the FLP/FRT system in the hybrid plants.
Impacts
Crop plants stably expressing FLP recombinase and use of a functional FLP/FRT recombination system in crop species will facilitate future biotechnology based applications for genetic modification of food crops through gene transfer strategies. Site-specific inactivation or removal as well as integration of transgenes, will improve our ability to manipulate plant genomes for trait enhancement in agriculturally important food crops in the United States.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Funds for this project were released in early December, 2001. The PIs have begun to address Objective 1 of this project. This involves assembly of two pSB11-based Agrobacterium binary vectors for transformation of rice. One vector contains a chimeric FLP recombinase and the second vector contains a recombination-reporter gusA gene. Using these two constructs, we will produce separate transgenic rice lines expressing FLP recombinase or containing the recombination-reporter gusA gene via Agrobacterium-mediated transformation in the following year. This vector construction phase of the project is nearing completion.
Impacts
Creation of crop plants stably expressing FLP recombinase and studying the functional efficacy of the FLP/FRT recombination system in crop species will facilitate the future use of this system in site-specific inactivation or removal as well as integration of genes, and provide an independent system that can be used in conjunction with Cre/lox. This will improve our ability to manipulate plant genomes in a wide variety of crop species important to agriculture in the United States through plant genetic engineering.
Publications
- No publications reported this period
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