Functional Analysis And Characterization Of OsEF1 In Rice (Oryza Sativa L. ) Early Flowering Mutant

Rice (Oryza sativa L.), a short-day (SD) plant, is one of the most important crops, it is also an model of the monocot plant, with the complete sequences of rice available, subsequently, it is urgent to study on the functional analysis of the rice genome.The floral induction is determined by internal genetic factor and external environment signal in higher plant, and triggering flowering is a dynamic and complicated spatial pattern.The flowering time, often termed as heading time in grains, the flowering is an symbol of the transition from vegetative phase to reproductive phase, so the major objective has been set up for decades is that several new early flowering or photoperiod-insensitive varieties of rice are cultivated.Take advantage of the clone the gene which is related to regulation of the flowering, combined with the molecular genetics and transgenic technology, accerlate the breeding process,Studying the internal molecular mechanism of early flowering species, as well as the mutants were easily obtained and located candidate genes'site where led to mutation by T-DNA tagging technology or Ac/Ds system and Tos17 retrotransposon,etc, thus it is obvious to shorten the period for cloning gene, thus it is convenientuseful to speed the breeding.In my dissertation, based on the total 300 sterile and early flowering mutants screened by one million rice mutation library, we amplified the flanking sequences of the insertaion site through PCR-Walking strategy, and located the instertion sites of the candidate genes with bioinformatics analysis, in addition, in terms of the co-segregation analysis between phenotype and candidate genes, we further confirmed the function of candidate gene by complementation test.In long day (LD),the rice mutant Osef1 cause the flower nearly 30 days earlier than wild type plant, according to the co-segregation analysis and complementation test combined with bioinformatics, it is indicated that this Osef1 mutagenesis resulted from Tos17 sequences insertation at 3'UTR in OsEF1, and OsEF1 encoded an protein which is similar to protein that functions gametophores development in Physcomitrella patens under light conditions, and this OsEF1 protein product is localized to the nucleus by making use of onion epidermal by particle bombardment. The OsEF1 held the strong auto-activation effect in yeast cell , we constructed the mutual reverse vector which is comprised of the candidate interation protein as bait and OsEF1 as prey co-transform the AH109, and we confirmed that there is real interaction between OsEF1 and OSK4, what is more , we found that OSK3 and OsEF1 existed interaction during the yeast system analysis.The recombinant vector fused GST or His tag gene successfully expressed OsEF1,OSK3 and OSK4 in Transetta(DE3) induced with IPTG, Western Blot further tesfied them, which provided the experimental ground for further study.We performed the RT-PCR and Real-Time PCR to analyse the expression of genes, in LD, expression of OsEF1 in various tissues is different, and the OsEF1 expressed in a more level in leaf blade and shleath than other tissues. The Osef1 had no effect on the expression of the OsLhy and OsPRR which are circadian clock components, whereas, Hd1, a gene had an important role in photoperiod pathway, expressed in a rather low level. Meantime, hd1 had no effect on the expression of OsEF1,thus we concluded that Hd1 is upstream of OsEF1 and controlled positively by OsEF1.Ehd1, another gene controlled flowering independently of Hd1 , Ehd1 epression increased in mutant Osef1,this indicated OsEF1 might down-regulated the Ehd1 epression. To sum up, our result demonstrated that in LD, OsEF1 inhibit flowering in rice through both Hd1 and Ehd1 flowering pathways.