The Genes Cloning And Functional Analysis Of OsOFP Transcription Factor Family In Rice (Oryza Sativa L.)

Transcription factors, also called trans-acting elements, could activate or inhibitgenes transcription through interacting with cis-acting elements in the promoterregion of the genes. They play key roles in plant growth, development, morphogenesis,stress resistance and secondary metabolism. Ovate family proteins are plant-specifictranscription factor family, whose functions involved in the regulation of plant growthand development were reported in Arabidopsis and tomatoes. The rice genomeincludes31OsOFP genes, however, little is known about the molecularcharacteristics of OVATE-containing proteins in rice.In this study, the gene structures and the encoded protein sequence features ofOsOFP were analyzed by bioinformatics. The expression profiles of OsOFPs werestudied by quantitative real-time RT-PCR (qRT-PCR). Meanwhile, the subcellularlocalizations of OsOFP proteins were analyzed using the transient transformation ofArabidopsis protoplast. Some transgenetic rice plants overexpressing OsOFP geneswere obtained by agrobacterium-mediated genetic transformation. Further studies onthe function of OsOFP22were performed based on the strong phenotype displayingof its transgenic plants. The recombinant protein of OsOFP22was purified byprokaryotic expression system, laying the foundation for further study on thebiological function of OsOFP22. The main results in this study are as follows:A diagrammatic representation of the OsOFPs distribution on the ricechromosomes showed that the OsOFPs are scattered throughout the10chromosomesof rice, and their distribution is in form of unequality and cascade. Phylogeneticanalysis showed all the other OFPs in Arabidopsis and rice was further divided into4major subfamilies except AtOFP17. The gene structures were analyzed using theonline tool Gene Structure Display Server (GSDS). The results displayed that most ofOFPs are inclined to be intron poor in Arabidopsis, rice, maize and sorghum. Thissuggested that OFPs structure maybe occur introns loss in plant evolution. Theconservative OVATE domain exist in the C terminal of OsOFP protein, meanwhile,the unknown motif2and motif3were predicted in the OsOFP protein.Gene expression pattern provides important clues for investigating gene function. The expression profiles of31OsOFPs showed the tissue-specific, and exhibiteddifferent expression patterns in coleoptiles, young leaves, young roots, mature leaves,glumes, young panicles and grains in filling stage. Interestingly, most of OsOFPs wasmore highly expressed during the seed developmental stage than other stages ofdevelopment. The2-kb5′-upstream regions of the OsOFPs were investigated usingPlantCARE database. The31OsOFPs promoters included Skn-1motif, and some ofOsOFPs promoters contained GCN4motif, both Skn-1motif and GCN4motifbelongs to seed developmental cis-elements. These indicated that OsOFPs areinvolved in the regulation of growth and development in rice, especially in the seeddevelopment.Thirty one OsOFPs were cloned from rice variety “Nipponbare” and wererecombined into an entry vector pDONR using the BP-clonase. Furthermore,16OsOFPs were constructed into the expression vector VP64-pCambia1301, and9OsOFPs were constructed into the expression vector EAR-pCambia1301by LRreaction, then10VP64-pCambia1301vectors and6EAR-pCambia1301vectorscontained OsOFPs were transformed into rice by agrobacterium-mediated method.Following, screened490T0transgenetic seeds, some T1, T2and T3transgenetic seedshave been harvested.To confirm the subcellular localization of OsOFP family, the coding sequences ofOsOFPs were cloned into the destination vector (pENSG-YFP) which the N-terminusfused the yellow fluorescent protein (YFP) reporter gene by LR reaction, theYFP-OsOFPs were transformed into protoplasts from Arabidopsis mediated bypolyethylene glycol. The localization pattern showed that24OsOFPs were targeted inthe nucleus, and the others were targeted in the nucleus with granular spots. Theresults were, in general, consistent with the bioinformatics prediction of the OsOFPssubcellular localization with WoLF PSORT and CELLO programs.The transgenetic plants overexpressing OsOFP22showed strong phynotypes.Compared with wild type (WT), the OsOFP22overexpressed transgenic rice plantdemonstrated shortened internode length of culm, lamina joint bending, wider leaf，erect panicle, increased culm diameter, flowering delay. The seeds become moreround, shorter and thicker. Chlorophyll content in leaf was significantly higher thanwild type from filling stage to maturity stage. Overexpression of OsOFP22conferedmore sensitive reaction to GA and reduced sensitivity to BL in the transgenic plants. This results implied that OsOFP22gene may function in mediating the interaction ofGA and BR signal pathways in rice.OsOFP22RI-1、OsOFP22RI-2and OsOFP22RI-3vectors were constructed andtransformed into rice mediated by agrobacterium. OsOFP22RI-1、OsOFP22RI-2andOsOFP22RI-3transgenic plants were obtained. The qRT-PCR results displayedOsOFP22RI-1vector did not inhibit OsOFP22expression, while both OsOFP22RI-2and OsOFP22RI-3could reduce OsOFP22expression in the transgenetic plants.There were no significant difference in height, tiller number, panicle internodeelongation, grain length and width, unhulled seed thickness, seed weight, leaf lengthand width between OsOFP22RI transgenetic plants and the wild type plants. Theseresults indicated that OsOFP genes may function redundantly in rice developmentregulation.To further investigate the properties and function of OsOFP22protein, OsOFP22was constructed into prokaryotic expression vector pCold TF which can be expressedinto E.Coli Transetta (DE3). The recombinant protein was induced expression andpurified from E.Coli Transetta (DE3) with Ni-NTAAgarose. These results contributeto the study on the properties of OsOFP22protein, cis-element which combined withOsOFP22protein and its target genes.