Transformation Of Ds Into Rice And The Transposition Analysis In Hybrid Descendants

Transposon tagging is an important tool for the study of functional genomics. By constructing the binary vector CamDs and transformation mediated by Agrobacterium, we developed an Ac/Ds transposon system that could be used for both gene trapping and activation tagging. The following results are obtained :1. Through Agrobacterium-mediated transformation, 62 independent fertile transgenic rice lines were produced.2. In T1 generations, 92.8% Ac lines and 93.5% Ds lines were identified as transgenic plants by PCR analysis. Southern blotting analysis for 32 independent transgenic plants indicated that the maize transposable element Ac/Ds had been integrated into the rice genome and most integration was one or two insertion sites.3. Resistance assay was applied to TO, T2 transgenic Ds rice and TO, T1 transgenic Ac rice. The resistant plants were planted in field. In order to induce transposition of the inserted Ds elements, the Ds-inserted transgenic plants were crossed with the transgenic plants carrying AcTPase. A population of about 290 hybrids consisting of Ac × Ds and Ds × Ac was constructed in this study.4. For the F1 population of Ac × Ds crossing offspring, 108 single hybrid plants of 12 combinations were studied. First, the hybrids consisting of both Ac and Ds were screened by hygromycin and kanamycin. Second, we could estimate that Ds were excised based on the Basta-resistance analysis. Third, the excision frequency of Ds element was evaluated with PCR amplification. If Ds element did not excise, PCR amplification would fail because of the long distance (8.3kb) between the two primers. Otherwise, a 860bp DNA fragment would be amplified. The result indicated that the excision frequency of Ds element trans-activated by Ac transposase was from 0 ~ 50% and the average excision frequency was about 13%.5. To assess the efficiency of gene trapping using the gus reporter gene, weexamined GUS expression patterns in various organs of F1 hybrids in different growth periods. The hybrid plant A4-1 displayed GUS expression in flowers and leaves at the flowering stage. This result implied that the gene trap system was able to identify the specific organs that were GUS positive at specific stage.6. Of transgenic TO lines and T] generations, some plants showed morphological aberrations, including dense spike (Ds31-l), yellow leaf (Ds147), slender (Ds9), albino, stripe, early tillering, growth promotion, leaf with big angle. Further molecular analysis is in necessity to testify whether these morphological aberrations are linked to T-DNA insertion or not.