TRANSACTIVATION AND STABLE INTEGRATION OF THE MAIZE TRANSPOSABLE ELEMENT DS-COTRANSFECTED WITH THE AC-TRANSPOSASE GENE IN TRANSGENIC RICE PLANTS

To develop an efficient gene tagging system in rice, a plasmid was con structed carrying a non-autonomous maize Ds element in the untranslate d leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid conta ining the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-res istant calli, regenerated plants and their progeny showed that the int roduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hyg romycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alteration s are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 bp target duplications is obse rved at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated st ably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stabl y integrated into three different chromosomes, one of which also conta ined the functional hph gene restored by Ds excision. The results indi cate that a dispersed distribution of Ds throughout genomes not bearin g the active Ac transposase gene can be achieved by simultaneous trans fection with Ds and the Ac transposase gene.