Molecular Analysis Of OsIRL Gene Family And Functional Study On Flowering Time Gene RID1

Rice is an important crop as well as a model for genome research of monocot plants. With the completion of rice genome sequencing project, emphasis on genomics is to determine the functions of the huge number of genes predicted by sequence analysis. Insertional mutagenesis is a high-throughout method for functional genomics, and the flanking sequence tag (FST) information and phenotypic data of mutant population are important resources for forward and reverse genetics studies. In this study, PCR tests were firstly performed for 15,494 independent transformants from our T-DNA insertional mutant library, and 88.3% of them were PCR-positive. Using the TAIL-PCR method, a total of 6,798 FSTs were isolated from the T-DNA insertional mutant population, among which 2,643 had significant similarity (E-value<10-5) with the rice genome sequences. In addition,277 mutant lines with obvious abnormal phenotypes were obtained by a large-scale phenotypic screening. Of 33 mutant lines examined in this study, the mutant phenotypes of 2 lines (04Z11EM13 and 03Z11DQ39) were confirmed to be cosegregated with their corresponding T-DNA insertions, respectively.The mutant phenotype of Line 03Z11DQ39 (osirl1) exhibited semi-dwarf and late-flowering, and the corresponding T-DNA tagged gene was named as OsIRL1 (Oryza sativa intracellular Ras-group-related LRR 1), a rice homologue of PIRL genes in Arabidopsis. Further investigation showed that osirl1 mutants showed a delay in flowering under both short-day (SD) and long-day (LD) conditions. The transcript levels of Ehd1 and Hd3a were largely reduced in osirl1 mutants, and Hd1 was partly repressed by osirl1 mutation under both SD and LD.In addition, we also found the expression of a clock-controlled gene Cab1R was also partly reduced in osirl1 mutants. However, the OsIRL1 transcript level did not seem to show an obvious discrepancy between wild types and osirll mutants under both SD and LD. Also, suppress expression or over-expression of OsIRL1 in transgenic plants did not cause a phenotype mimicing the same or opposite phenotype of osirl1 mutants. Thus, it seems that the osirl1 mutant phenotype was not controlled by OsIRL1.Compared with all previously identified plant LRR proteins, Arabidopsis PIRL proteins are more closely related to Ras-group LRR proteins that can interact directly with Ras GTPases and take part in signal transduction in animals and yeast. Except OsIRL1, another 7 rice homologs of PIRL proteins were also idntified as members of OsIRL protein family in this study. We described the gene structures, chromosome localizations, protein motifs constitution, and phylogenetic relationship of the OsIRL gene family. Most of OsIRL genes were found to be located within the duplicated segments of the rice genome with none found being organized in tandem clusters. The expression profiles of OsIRL genes have been analysed under entire rice developmental stages, along with light and three hormones (GA, NAA and KT) stress conditions using quantitative RT-PCR and microarray data mining. All OsIRL genes were expressed in at least one of exprimental stages studied and exhibited divergent expression patterns, with several genes showing preferential expression at specific stages. Differential expression of five OsIRL genes under stress conditions was also evident and four genes responded to two or more stress treatments, suggesting that they may be involved in the interactions of light and hormone signals at the physiological level. We also reported the collection, identification and characterization of 5 T-DNA or Tos17 insertion lines for 4 OsIRL genes using the reverse-genetics strategy. A comprehensive expression profiling undertaken in this investigation together with the characterized insertion lines will provide a solid foundation for in-depth dissection of the functions of OsIRL genes.Precise control of flowering time (or heading date) is necessary for successful sexual reproduction in plants. Molecular-genetic analysis of flowering time mutants have been used as an effectual strategy to identify genetic pathways and regulatory proteins associated with the control of flowering time. Previous studies have identified a never-flowering rice mutant (rid1) caused by a T-DNA insertion at the RID1 (Rice Indeterminate 1) locus. In this study, the full length cDNA sequence of RID1 was obtained by rapid amplification of the cDNA ends (RACE) and RT-PCR amplification. The nuclear locaization of RED1 protein was confirmed by a transient expression assay in onion epidermal cell. The results of quantitative RT-PCR analyses showed that the express of Ehd1, Hd3a and RFT1 was nearly completely repressed by rid1 mutation, and the expression of Hd1 and Ghd7 was also partly affected by RID1 under both SD and LD. Apart from Hd3a and RFT1, mRNA levels of five addition FT-like (FTL) genes examined in this study were also affected by rid1 mutation. In addition, RID1 seems to be independent of the circadian clock. A model was proposed to place RID1 in the molecular pathways of flowering regulation in rice, for which there are two indispensable elements. In the first, RID1 acts as a upstream flowering promoter controlling two independent floral pathways (Hd1-dependent and Ehd1-dependent pathways) as well as other unknown pathways. In the other, Hd3a/RFT1/FTL complex acts as a downstream integrator of flowering-time signals from different pathways and thus induce flowering. Completely abolishing the function of either element would cause never-flowering in rice.