Date of Graduation

12-2012

Document Type

Thesis

Degree Name

Master of Science in Crop, Soil & Environmental Sciences (MS)

Degree Level

Graduate

Department

Crop, Soil & Environmental Sciences

Advisor/Mentor

Vibha Srivastava

Committee Member

Ioannis E. Tzanetakis

Second Committee Member

Mary C. Savin

Third Committee Member

Nilda R. Burgos

Keywords

Biological sciences, Flp/frt, Site-specific recombination, Transgenic

Abstract

Site-specific recombination systems are powerful tools for genetic modification. They have been used to integrate a transgene into a pre-defined locus and to remove marker genes from a transgene locus. Two of the most widely used site-specific recombination systems in plants are the Cre/lox system from the bacteriophage P1 and the FLP/FRT system from the yeast Saccharomyces cerevisiae. The Cre/lox system is well-characterized and is the first choice in application of site-specific recombination system. However, some applications such as marker-free site-specific gene integration require the use of two recombination systems. In addition, the availability of alternative recombination systems can offer a flexible choice or the opportunity to develop multiple applications in a single platform. Hence, the FLP/FRT system should be evaluated further for its recombination efficiency, particularly in rice, a model crop plant. Some studies using FLP/FRT systems, with the wild type FLP called FLPwt recombinase, reported low efficiency for regular application of the system in removal of transgenic locus. However, two improved versions of FLPwt: FLPe (thermostable version of FLPwt) and FLPo (mouse-codon optimized version of FLPe) are available and have not been carefully tested in plants.

To look for the best choice of FLP recombinase variant in the application of the FLP/FRT system in crop genetic engineering, the relative recombination efficiencies of FLPwt, FLPe and FLPo for marker gene excision from the transgene locus in rice were evaluated. FLPwt, FLPe, and FLPo transgenic rice lines were generated and FLP activity in these lines was evaluated. These experiments revealed that FLPe and FLPo had much higher activity than FLPwt in removing FRT-flanked npt segment to fuse GUS gene with the promoter. These experiments also indicated that FLPo is relatively more efficient than FLPe. Thus, based upon results from the present study, I recommend the use of FLPo in plant genetic engineering.

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