Functional Analysis Of Rice Flowering OsEF1 Gene

Rice (Oryza sativa L.) is one of the most important crops in the world, and also regarded as the model plant in the studies of grass family and monocotyledon. With the completion of rice genome sequencing and establishment of a large number of rice mutant libraries, the pace of theoretical studies of rice gene was accelerated greatly and the period of cloning one gene was shortened, the rice genome research went into the post-genome era. Recently, a lot of important agronomic traits were successfully cloned, such as florescence, sterile, leaf shapes and disease resistance and so on. Flowering, also known as heading time in rice, is a transition from vegetative growth to reproductive growth. Therefore, cloning of rice flowering related gene would help to understand the molecular basis of regulatory network and improve the control of flowering in rice, expand the area of rice cultivation, and provide important genetic basis and theoretical guidance for the breeding.This article is about an early flowering mutant Osef1 which was screened from the one million mutant library of our laboratory. This work is based on the following results: According to the flanking sequence amplification result and complementation test, we proved that the Osef1 phenotype was resulted from Tos17 insertion at 3'UTR in OsEF1gene. A nuclear green fluorescence after transient expression of OsEF1 fused to GFP in onion epidermal cells revealed that OsEF1 gene was localized in the nucleus. We construct the confusion vector pBDGAL4cam-EF1, used yeast two-hybrid method to acquire thirty-five candidate proteins which interacted with OsEF1 protein. We also proved that OsEF1 positively regulated the expression of Hd1 and negatively regulated the expression of Ehd1 in the long-day condition.Firstly, the field survey of agronomic traits found that the flowering time of Osef1 mutant is 30day earlier than the wild-type in the natural long-day conditions. Also, the mutant showed semi-dwarf phenotype.Secondly, Expression pattern of OsEF1 gene were analyzed. Through the GUS staining in pP_OsEF1-GUS transgenic lines, we find OsEF1 expressed in root, stem, leaf, young spikelet, lateral root, node, leaf sheath, leaf joint. The GUS activities have been also detected in all types of leaves and leaf sheath by paraffin-embedded sections. It suggests that the OsEF1 acts as a constitutive expression gene in rice various tissue. The conclusion of Quantitative Real-Time PCR is consistence with the GUS staining result.Thirdly, the diurnal expression patterns of OsEF1 were analyzed in wild type under short-day condition by quantitative RT-PCR. Ubq gene was selected as internal control. We found that OsEF1 was expressed throughout the light/dark cycle, with a peak at the beginning of the light. The result suggested that OsEF1 transcription was controlled by circadian clocks and exhibited a diurnal rhythm expression pattern under SD conditions.Fourthly, in order to better research the target of OsEF1, RNAi suppression method was used to inference the expression of the eight genes which were screened by yeast two-hybrid system. Mainly use the Gateway system or pTCK309 vector to construct the RNAi vectors. Among them, they are seven vectors which are constructed by Gateway system and the other by pTCK309 vector. The construction vector was transformed to Nipponbare background by Agrobacterium-mediated method. Then there are three lines with early flowering in one of the eight transgenic groups.Lastly, the full-length cDNA of OsEF1 was fused on PET-32A vector. This confusion vector can express soluble protein successfully in the 0.4mM IPTG under 8 hour induction. Based on the detection of His-tagged protein by Western Blot, the expressed total protein is the target protein. HIS nickel column purified this protein by a small amount under native conditions and found 150mM imidazole was more appropriate. The large-scale purified protein was used for the preparation of polyclonal antibody to further detect endogenous protein of Osef1 mutant. Detailed functional analysis and characterization of OsEF1 and compared with Arabidopsis, we find that only the unique and complex gene pathway could provide rice plants the facultative short-day nature so that they can also flower during non-inductive conditions.