Construction Of Rice Mutant Library Using Ac/Ds Tagging System And Its Application

With the completion of the rice genome sequence, the rice functional genomic study has become the major task of rice molecular biological research. Developing of rice mutant library is an essential approach for rice functional genomic study. Utilizing Ac/Ds (Activator/Dissociation) tagging system is a promising method to construct rice insertional mutagenesis library. Transposon tagging is a strategy employed to generate insertional mutation in higher plants, by which the mutanted genes can be easily identified by isolating the DNA flanking sequences of the inserted transposon. In this research, we have selected japonica cultivar Yandao 8 as the acceptor variety, and constructed the transposon insertion mutant library of rice by using transposable element system. The major results are as follows:1. The transposon Ac and Ds had been transferred into Yandao 8 genome, respectively, by Agrobacterium tumefaciens mediated transformation, and a total of 215 Ds transformants and 148 Ac transformants were generated.2. By PCR analysis, Hygromcin resistant detection, as well as GFP screening, 130 Ac positive plants and 208 Ds positive plants were obtained. The positive rates of the transformed plants are 87.8% and 96.7%, respectively.3. In T1 generation, 18 kinds of mutants were found. Among them, eight mutants were confirmed to be resulted from T-DNA insertion. The mutated genes in six mutants were further isolated by the flanking sequence of the inserted transposons. 4. We selected 44 Ac and 72 Ds positive plants as the starter lines of crosses, respectively. They are with single copy of transgene but without any altered phenotypes.5. 16 plants haboring Ac and Ds in same plant were detected in the F1 generation by PCR analysis. Those self-pollinated F1 plants gave rise to F2 families. To identify putative transposants, DSE-PCR ,DSC-PCR, Tail-PCR and GUS detection were performed on F2 lines. An average of 50%of the F2 families were found to have germinal transpositions among 16 lines.6. By investigation of the F2 mutant phenotype, we found five lines have altered phenotypes. The mutanted genes in the mutants were further identified by tranposon tagging.