Function of Cre/loxsite-specific recombination system in the rice genome and its implication

Abstract

The Cre/lox site-specific recombination system was used to excise a transgene from the rice genome. Transgenic rice plants containing hygromycin phosphotransferase gene hpt flanked by two lox sites and those containing the Cre gene were developed through Agrobacterium-mediated transformation. In the hybrids containing both lox/hpt/lox and Cre, the hpt gene was excised because of site-specific recombination. The excision could be detected because it caused the fusion of the promoter of the hpt gene with a promoterless distally located gusA gene. As a result, the gusA gene became active and its expression was visualized. With this strategy, T 2 lox/hpt/lox and T 2 Cre transgenic plants were crossed. Out of 132 hybrid plants from 12 crosses, 77 plants expressed GUS+ , of which 32 plants showed the hpt gene to be excised completely and 45 plants had the original band excised but exhibited more than one band as recombined products. GUS+ expression was found only in the hybrids and not in the parental lines or their selfed progenies as well as untransformed controls, an indication that the excision event had occurred only in the hybrid plants. Site-specific recombination systems have been found to be functional in a variety of higher eukaryotes, including plants and mammals. Of the four site-specific recombination systems, Cre/lox has been the most highly characterized in plants. With this system, site-specific recombination is directed by the Cre protein, which recognizes and interacts with two lox sites to direct excision, integration, or inversion of the intervening DNA sequences, depending on the orientation of lox sites. The function of the Cre/lox site-specific recombination system to excise a DNA sequence from the genome of yeast and mammals was reported earlier (Sauer 1987). Later, this system was used to excise a selectable marker from the host genome of tobacco (Bayley et al 1992) and Arabidopsis (Russell et al 1992). In our study, we examined the function of the Cre/lox system in the rice genome. Transgenic plants containing lox/ hpt/lox/gusA or Cre/hpt were developed. We demonstrated that, when the Cre gene and lox sites were brought together into the same hybrid plants by crossing, the hpt gene flanked by the lox sites was excised effectively from the genome because of site-specific recombination of lox sites catalyzed by Cre recombinase.