Generation Of T-DNA Insertional Mutants Library And Isolation And Characterization Of OsLIS-L1Is Reqyired For Plant Height And Male Gametophyte Formation In Rice

Rice is a very important food crop and model plant of monocotyledons for functional genomics research, about half the world's population with rice as staple food. After completed sequencing of many species including rice genomes, we have started the most key step for functional genomics, thus generation large-scale T-DNA insertion mutant library is a very important technical platform for functional gene research on rice whole genome. In this study, we participated in the generation T-DNA insertion mutant library, and generated about60,000independent T-DNA insertional mutants with workers.Plant height is an important component of grain yield that is related to lodging resistance and fertilizer endurance. Dwarf mutants have led to'green revolution'in the1960s through the development of semi-dwarf, high-yielding varieties of rice, making the rice yield have an unprecedented breakthrough. Henceforward, the basic genetic research of the plant height was widely studied. The development of plant anther is a very complicated process, it includes the development of pollen sac wall, pollen mother cell, microsporogenesis and mature pollen grain, so the fertility can be affected by any of these abnormal development. Male gametophytes development is a very complicated process, which is related to many genes. It has the theoretical and practical values by studying the physiological and biochemical functions of these genes. Here, we screened3,905mutant lines and getting134preliminary co-segregated lines showed abnormal phenotype and1co-segregated line, then we isolated a new gene OsLIS-L1, which controls plant height and male gametophyte formation in rice. The main study results were described as follows:1. Generating independent T-DNA insertional mutants about60,000with workers.2. Screening3,905mutant lines and getting134preliminary co-segregated lines showed abnormal phenotype and1co-segregated line:03Z11AI23(Chen Zhihui in our lab provide this mutant), the semi-dwarf and very low fertility mutant segregated from03Z11AI23was designed as oslis-l1-1, more detail morphological characterization was done with the oslis-l1-1mutant and isolate and character the corresponsive gene OsLIS-L1. Co-segregation analysis in T1and T2generation of03Z11AI23showed that the mutant phenotype was co-segregated with the T-DNA homologous insertion. 3. We ordered an allelic mutant3A-04974which was designated as oslis-l1-2, from POSTECH, South Korea. The mutant oslis-l1-2also showed semi-dwarf and very low fertility, the genotyping of oslis-l1-2was confirmed and the mutant phenotypes co-segregated well with the T-DNA insertion.4. OsLIS-L1RNAi suppressed lines in WT background exhibited semi-dwarf and very low fertility, which mimicked the same defects with the two allelic T-DNA insertional mutants. And transformation with the genomic fragment containing the entire OsLIS-L1gene to oslis-l1-2mutant callus homozygous yielded the WT phenotype. Taken together, these results confirmed that semi-dwarf and very low fertility of mutants was caused by the loss of OsLIS-L1function.5. Different truncated OsLIS-L1transcripts generated in the two oslis-l1mutants by RT-PCR analysis.6. We observed oslis-l1-2mutant chromosomal behavior during the meiosis process of the pollen mother cells and find that there were no obvious differences between oslis-l1-2and WT, suggesting that the oslis-l1mutations have not affected chromosomal behaviour during meiosis in pollen mother cells.7. The mutant pollen failed to stain with KI-I2indicated that the mutant was male sterility. Pollinating the flowers from oslis-l1-2with WT pollen can normally produce seeds indicated the mutant was almost female fertility.8. We compared the different development stages of the anthers in the WT and oslis-l1-2mutant plants by histological examination of plastic transverse sections. No obvious differences of anther cell wall, microspore mother cell and microspores development were observed between oslis-l1-2and WT, but from stage10to mature pollen stage, in the oslis-l1-2anther, almost pollen grains collapsed and had no nutrients in the cytoplasm. These findings suggest that the low fertility in oslis-l1might be due to the failure of normal microspores formation. Consistent with the analysis for semi-thin sections of anthers, no obvious morphological changes were observed between anthers of the WT and oslis-l1-2by TEM, the middle layer and the tapetum are normally degenerated, and the pollen wall development was also normal.9. We compared the length of five internodes from the main culms between the WT and oslis-l1-2mutant plants respectively. Interestingly, we found that the length of the first internodes under panicle differed most between WT (32.1cm) and oslis-l1-2mutant plants (22.1cm), but there were no distinct differences between the other corresponding internodes length comparisons. Next, plastic transverse and longitudinal sections were employed to histologically analyze the first internodes structure of the WT and oslis-l1-2mutant. The results showed that the stems of oslis-l1-2mutant plants consisted of the same layers and cell size with the WT, suggesting that the decreased height of the stems in oslis-l1plants is mostly due to decreased cell proliferation rather than cell size in the first internodes.10. Quantitative RT-PCR analysis showed:OsLIS-L1transcript had a relatively higher abundance in stem and panicles at the heading stage, though other tissues at the same stage such as root, leaf and sheath also showed weak expression of OsLIS-L1. This expression pattern of OsLIS-L1supported its function for internode elongation and panicle development in rice. Subcelluar localization analysis suggests that OsLIS-L1is a nuclear protein.11. We identified two OsLIS-L1ortholog in rice.