Development Of A Two-element Ac/Ds Transposon Tagging System In Rice Using Fluorescent Proteins

With the completion of rice genome sequencing, rice functional genomics has become an important field of rice research. Mutagenesis is an important method for discovery of new genes and characterization of gene functions. The transpo son system is one of important approach for construction of insertional library of the rice genome. In this study, a two-element Ac/Ds transposon tagging system was developed using the green fluorescent protein (GFP) and red fluorescent protein (RFP), and Ac transposase (AcTPase) and Ds element were transformed into the japonica rice Zhe04B respectively. Genetic analysis of F1and F2generations was performed using the fluorescent visual markers. Research results were obtained as follows:1. In T1generation,64.29%of the Ac transgenic plants and65.71%of Ds transgenic plants segregated in the3:1Mendelian genetic pattern, indicating that the Ac-T-DNA and Ds-T-DNA in more than60%of transgenic rice lines integrated at single locus of the rice genome. Based on TAIL-PCR results, the T-DNAs of Ds-1and Ac-46transgenic rice lines were inserted into chromosome1(Chr01:42,099,744) and chromosome5(Chr05:20,527,399), respectively.2. PCR analysis of the empty donor site of Ds transposable elements (EDS-PCR) showed that an average of75%(Ac/Ds) F1plants underwent somatic transposition. Ds excision footprints derived from independent transposition events were detected in different F1plants and different cells in the same plant. Semi-quantitative PCR of rice genomic DNA of (Ac-46/Ds-1) F1and (Ac-46/Ds-35) F1plants was conducted, and the relative content of EDS and FDS indicated that transposition in (Ac-46/Ds-1) F1plants occurred earlier than in (Ac-46/Ds-35) F1plants. Semi-quantitative RT-PCR of AcTPase showed that the AcTPase expression levels in (Ac-46/Ds-1) F1was higher than that in(Ac-46/Ds-35) F1 3. GFP and RFP fluorescent of pollens of the (Ac-46/Ds-1) F1plants were observed with Confocal microscope.30.6%of the pollens showed GFP positive and RFP negative (GFP+/RFP-), suggesting that transposed Ds element segregated from Ac and became genetically stable. Therefore, high-frequency transposition events occured in germ cells of F1plants.4. RFP and GFP were utilized as negative and positive selectable markers, respectively, for genetic analysis and screening of27(Ac-46/Ds-1) F2populations. Genetically stable transposants (i.e. GFP+/RFP-plants) were obtained from59.3%(16/27) of the F2populations, because11.1%(1/9) of the (Ac-46/Ds-35) F2populations segregated stable transposants (GFP+/RFP-).5. TAIL-PCR analysis of stable transposants was performed for isolation of Ds flanking sequence tag(FST). Ds in a part of the transposants integrated into the genie regions of the rice genome.In summary, more than50%(Ac/Ds) F1plants produced at least a stable Ds transposition plant in F2generation. The Ac/Ds transposable element vector system described in this study can be effectively applied for construction of large-scale insertion mutant library of rice as well as of other plants of the grass family.