| By constructing three binary vectors (pDsE, pDsG and pAc) and T-DNA transformation mediated by Agrobacterium, we developed an Ac/Ds system harboring gene trap and enhancer trap for gene tagging in rice. The following results are obtained:1. Through Agrobacterium-mediated T-DNA transformation, 1188 independent fertile transgenic rice lines were produced. Injaponica Nipponbare, 526 lines were obtained including 51 NAc lines transfromed with pAc, 361 NE lines transfromed with pDsE and 114 NG lines transfromed with pDsG Injaponica Zhonghua-11, 662 lines were obtained including 176 CAc lines transformed with pAc, 320 CE lines transformed with pDsE and 166 CG lines transformed with pDsG Hygromycin resistance tests revealed that transgenic plants contain an average of 1.3 loci of T-DNA inserts. Southern blot analysis of transgenic plants indicated that they contain average 1.8 T-DNA copies in rice genome.2. The distribution of GC content of rice gene region inserted by T-DNA was very similar with that of rice genome DNA. Through GC content analysis of SOObp rice genomic DNA flanking insertion site in 126 lines, we found that the average GC content of the insertion site was 42.5% and 83% of the T-DNA insertion lines locate in a genomic domain ranging from 30% to 50% in terms of GC content. Fifty (40%) out of the total 126 T-DNA insertion lines were in the gene region of rice.3. Both precise insertion and imprecise insertion existed when T-DNA integrated into rice genomic DNA. Among total 182 T-DNA/rice DNA junctions, only 69 (38%) were found with no filler-DNA, whereas the remaining 113 (62%) contained filler-DNA from Ibp to several hundred base pairs. By analysis of 61 filler-DNA larger than 50bp, we found that 49 (80%) were from the sequence within T-DNA. Eight (13%) of them were from backbone sequence of binary vector out of T-DNA. In addition, 4 (7%) had no similarity with rice or vector sequences. Three signature nucleotides TGA of right border repeat were kept in 77 out of 158 T-DNA insertions. No specific nucleotide where the T-DNA left border broken was found when T-DNA integrated into rice genomic DNA. Microhomologous fragments from 1 to 8bp were observed in the sequences nextto the left border of T-DNA insertions. Sixteen direct ligation of T-DNA right border to left border were observed in our experiments.4. The structure of genomic DNA at insertion site affected Ds transposition frequency greatly. Ds excision frequency varied from 0% to 40% in 20 F2 populations derived from 11 different Ds parents with PCR analysis. It was found that Ds copy number had little influence on Ds transposition frequency. Southern blot analysis revealed that more than 70% of Ds element reinserted into rice genomic DNA and most of them (>70%) were independent in F2 population. 14-33% of Ds reinsertion FI plants were stable without Ac element.5. Ds is inclined to integrate into gene region of rice genomic DNA. Seventeen out of total 29 (59%) flanking sequences of Ds elements were identified to be within gene region of rice genomic DNA, including 6 in promoter, 4 in exon and 7 in intron. Another 10 flanking sequences of Ds elements were proved to be within intergenic regions. 8bp duplication of rice genomic DNA appeared at insertion site of Ds element.6. Among the F2 plants with independent Ds insertions, approximately 28% of enhancer trap and 22% of gene trap inserts displayed GUS activity in leave, roots, flowers and seeds.
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