| The creation of insertional mutant library and isolation of the flanking sequence tags (FST) of the insertion tags to construct an indexed FST database is one of the important strategies for gene cloning. In this research, we isolated 7035 FSTs from our T-DNA insetional mutant library by TAIL-PCR, which enciched the mutant FST source and will benefit the reverse genetics strategy in rice gene cloning.Meiosis is a central step during the sexual reproduction in eukaryotes and is essential for their metagenesis. Over the past decade with yeast and Arabidopsis leading the foray, we have witnessed an explosion in understanding of the mechanisms of the meiosis process; however, we still know little about the meiosis control mechanism in rice, quite a disproportion to its important status in both agricultural production and academic research. In this research, we conducted the functional analysis of OsRPA1a and OsRPA2-3, which participate in the meiosis process in rice, exploring two sterile mutants generated by T-DNA insertion; the main results are as follow:1. Both of osrpa1a and osrpa2-3 mutants are identical to wild type in plant morphology, but show sterile phenotype at mature stage. In addition, suppression of OsRPA1a or OsRPA2-3 expression in wild type phenocopies the osrpa1a or osrpa2-3 mutation, respectively. And transformation of callus homozygous for the mutants with the genomic fragment containing the entire OsRPA1a or OsRPA2-3 gene yielded the wild-type phenotype, respectively.. Taken together, these results confirmed that the sterility of mutants was caused by the loss of OsRPA1a or OsRPA2-3 function.2. Osrpa1a and osrpa2-3 mutants have the same defects in female gametophyte development at the megasporocyte meiosis stage, resulted the abnormal functional megaspore. At mature stage, the structure of the typical "seven cells and eight nuclei" in the wild type embryo sac were instead of the footprint of degenerated nucleate in osrpa1a and osrpa2-3 mutants.3. In osrpa1a mutants, homologous chromosomes pairing, synapsis and forming normal 12 bivalents in prophaseâ… as that of wild type, however, chromosome fragment presents in the pollen mother cells after metaphaseâ… , which indicates there still exists unrepaired DNA damage in the PMCs in osrpa1a mutants.4. In osrpa2-3 mutants, homologous chromosomes achieve pairing and synapsis normally, but the formation of chiasmat decreased dramatically, leading to the presence of univalents at diakinesis. In contrast to osrpa1a mutants, osrpa2-3 mutants do not show chromosome fragment during the meiosis process.5. Compared with wild type plants, osrpa1a but not osrpa2-3 mutants show hypersensitivity to MMC, MMS and UV irradiation, which indicates the different requirement of OsRPA1a and OsRPA2-3 in DNA repair pathway.6. After treated with MMC and UV, there is more unrepaired DNA damage presents in osrpa1a mutants, moreover, the unrepaired DNA damage presents earlier than in wild type.7. Mitosis is unaffected in osrpa1a and osrpa2-3 mutants, which suggests that OsRPA1a and OsRPA2-3 may not participate in, or at least dispensable for, DNA replication in rice. |
