Functions And Molecular Regulatory Mechanisms Of OsFBH Gene Family In Rice

Flowering time plays important roles in development and formation of rice(Oryza sativa).Flowering time of rice is controlled by endogenous and external factors,which is involved in multiple genes.Although the regulatory network of flowering time control has been established in which Heading date 1 and Early heading date 1 act as key factors,the molecular mechanisms on regulation of flowering time are far from known.In this study,OsFBHs were selected based on homology and phylogenetic analysis in order to analyze the function of Arabidopsis(Arabidopsis thaniala)FBH homologs in rice,termed OsFBHs.CRISPR-Cas9,recombinant DNA and transgenic technology were applied to generate overexpression,RNA interference,and gene editing transgenic lines of OsFBHs in rice and Arabidopsis.According to the result of phenotyping,functional genes were chosen.By using modern molecular biotechnology,such as real-time quantitative RT-PCR,GUS-staining,yeast one-hybrid,yeast two-hybrid,and Chromatin Immunoprecipitation(CHIP),we study the OsFBH expression pattern,subcellular location,target gene so as to reveal the molecular mechanism of OsFBHs regulating rice heading date.Our research achievement will provide scientific evidence to production and quality improvement in rice.The major results of our experiment are as follow.1.OsFBHs from rice is phylogenetically conserved to FBHs.Results of phylogenetic analysis showed that OsFBH1 and OsFBH2 are conserved with FBH1 and FBH2;OsFBH3 and OsFBH4 are conserved with FBH3 and FBH4.In addition,both OsFBH and FBH proteins contain a conserved HLH domain..2.Expression partern and subcellular localization of OsFBHl in rice.Results of real-time quantitative RT-PCR and GUS staining assay showed that OsFBH1 expressed mainly in leaf and sheath.A peak expression of OsFBH1 at 50 days after sowing was observed.OsFBHl expression is regulated by circadian clock;the expression peak occurred at night.In addition,OsFBH1 protein was localized in the nucleus.3.Overexpression of OsFBHl delayed the flowering time in Arabidopsis.Comparing with the wild type(WT,Columbia-0),rosette leaf number was increased;the days to the flower bud emerging and first flower opening were significantly delayed by overexpression of OsFBH1 transgene in Arabidopsis.The results of developmental phase transition assay showed that overexpression of OsFBH1 could increase the number of juvenile leaf and adult leaf,which expand transition time from vegetative growth phase to reproductive stage in Arabidopsis.The expression of Flowering locus T(FT),Twin Sister of FT,Suppressor of Overexpression of CONSTANS 1(SOC1)was reduced in Arabidopsis OsFBHl overexpression transgenic lines,which delayed the flowering time in Arabidopsis.4.Overexpression of OsFBH1 delayed the flowering time of rice.Comparing with wild type(SN9816),heading date was delayed in overexpression of OsFBH1 transgenic lines.The results of real-time RT-PCR showed that overexpression of OsFBH1 suppresses the expression of flowering time key regulators,such as Heading date 3a and Rice Flowering locus T 1,resulting in late flowering of rice.In addition,Overexpression of OsFBHl reduced the plant height,the width of grain,and the number of tillers.5.Overexpression of OsFBH3 caused early flowering under both long day and short day conditions in Arabidopsis.Rosette leaf number was reduced;the days to the flower bud emerging and first flower opening were significantly accelerated by overexpression of OsFBH3 in Arabidopsis.The results of developmental phase transition assay showed that overexpression of OsFBH3 did not impact the vegetative phase transition,but accelerate vegetative to reproductive phase transiton by reducing the number of adult leaf to promote flowering in Arabidopsis.The expression of CONSTANS,FT,SOC1 was activated by overexpression of OsFBH3,causing early flowering phenotype under both long day and short day conditions in Arabidopsis.In addition,overexpression of OsFBH3 caused other developmental defects in Arabidopsis,includingsmall plant size,small leaves and flower,thin bolt,short siliques,reduced number of seeds per siliques and increased aborted embryos.6.Expression partem and subcellular localization of OsFBH3 in rice.Results of real-time quantitative RT-PCR and GUS staining assay showed that OsFBH3 expressed mainly in leaf,sheath,and stem.A peak expression of OsFBH3 at 50 days after sowing was observed.OsFBH3 expression is regulated by circadian clock;the expression peak occurred at night.In addition,OsFBH3 protein was localized in the nucleus.