Construction And Analysis Of A Novel Ac/Ds Tagging Mutant Library In Rice

With the completion of the rice genome map-based sequence in 2005, the study on functional genomics in rice has become a major task. Establishment of rice mutant library is an essential approach for rice functional genomics study. In this study, we constructed a large scale insertional mutant population using Ac-starter lines and Ds-T-DNA-containing anchor lines which could be used to select effectively and easily transposants at seedling stage, minimizing labor and time involved. We also systemically analyzed the transposition frequency in F1 and F2 generations as well as transpositional behaviors of Ac/Ds system. In addition, a large number of morphological mutants were generated in F2 and F3 lines, and primary analysis was carried out for produced mutants. The major results obtained are as follows:1. An insertional mutant population of Nipponbare (Oryza sativa L. ssp. japonica) was developed using a novel and efficient Ac/Ds tagging system. We verified reliability and feasibility of Basta selection in the field. The selected transposants by spraying Basta in the fields were in agreement with the results of PCR detection in the laboratory, which showed that this system was suitable to construct rice large-scale insertional mutagenesis library.2. A total of 636 lines containing 137,437 F2 plants were produced from 66 cross combinations derived from four Ds-starter lines and twenty Ac-starter lines, and there were 36,160 transposants among 137,437 F2 plants. The somatic transposition frequency was very high in Fl plants, but we failed to detect the germinal transposition in F1 plants. Among four Ds-starter lines, Ds1, Ds3 and Ds4 showed a high activity of transposition in F2 generation, and their germinal transposition frequency was 46.9%, 37.6% and 36.9% respectively. The germinal transposition frequency presented prodigious difference among different F2 siblings derived from the same cross combination, and this difference maybe resulted from difference of transposition timing. For Dsl, Ds3 and Ds4-starter lines, the germinal transposition frequency was mainly distributed within range of 30%-50%.3. Ds2 displayed a very low activity of transposition among four Ds-starter lines, only six out of 265 F2 families derived from Ds2-starter line showed transposition events at low frequency, and the rest of them displayed inactivation of Ds transposition. The reasons of inactivation of Ds transposition did not resulted from inactivation of AcTPase. We speculated that Ds inactivation was originated from Ds2-starter line.4. We ananlyzed the effect of cross combination patterns (Ds/Ac and Ac/Ds) and copy number of Ac-starter lines on germinal transposition frequency, showing that different cross combination patterns did not affect the germinal transposition frequency. And there was no significant difference for germinal transposition frequency based on Ac-starter lines between one and two-copies.5. Analysis of Ds flanking sequences showed that 52 flanking sequence tags (FSTs) were distributed on 10 rice chromosomes, and there were two FSTs near by its original T-DNA donor sites, and therest of them were far from its original T-DNA donor sites among 52 FSTs. There were Ds insertional clusters on chromosome 4 and chromosome 11, accounting for 54% of total FSTs. Moreover, 67.3% FSTs were found to insert within gene with predicted coding information of amino acids among 52 FSTs. FSTs of siblings from the same F2 line were positioned in different clones, even on different chromosomes, showing that siblings from the same F2 line could generated independent transposition events.6. We analyzed GUS activity in different organs of F3 transposants, showing that GUS positive was displayed in root, leaf, young panicle and floral organs. GUS activity was different for different insertional lines in different organs. Moreover, GUS activity was also different in different developmental stages. These results showed that gene trapper displayed strong trap function in insertional lines.7. A large number of morphological mutants were generated by our Ac/Ds system in F2 and F3 transposed lines. We investigated phenotype traits and agronomic traits about those mutants. The primary genetic analysis showed that cull5(t) mutant was regulated by single recessive nuclear gene, and its phenotype was co-segregated with Ds insertion. Cull5(t) mutant was sensitive for exogenous GA3.8. We analyzed insertion site of insertional line OsPI-PLC2 using reverse genetics method. RT-PCR results showed that OsPI-PLC2 gene was induced expression pattern, which could be activated by different environmental stress and chemical osmotic stress. BLAST results found that OsPI-PLC2 protein displayed higher identity with AtPI-PLC protein but lower identity with OsPI-PLCl protein.