The Functional Analysis Of OsOFP22 Transcription Factor

Ovate family proteins(OFPs)are plant specific transcription factors and are found universally to exist in monocots and dicotyledonous plants including Arabidopsis thaliana,pepper,tomato,banana and rice.OFPs regulate multiple aspects of plant growth and development like ovule and vascular bundle formation,fruit shape regulation,DNA repairing,second cell wall formation and fruit ripen.OFPs are reported to be involved in hormone anabolism and signal transduction of gibberellins(GA),brassinolides(BR)and Ethylene.Overexpression of At OFP1 in the transgenic Arabidopsis causes the leaves to be shorter and rounder,results in round seed pods,higher chlorophyll content and delayed flowering time.In Arabidopsis,At OFP1 functions as a transcription repressor.Rice plants over-expressing OsOFP2 showed altered leaf morphology and seed shapeis,and OsOFP2 is speculated to interact with KNOX and BELL classes transcription factors to inhabit the synthesis of the phytohormone GA.OsOFP8 ovexpression makes the lamina joint bending increased,contrarily,the lamina joint bending decreased in the OsOFP8 RNAi transgenic lines.Furthermore,Phosphorylation of OsOFP8 by Os GSK2 is needed for its nuclear export and the phospphorylated OsOFP8 shuttles to the cytoplasm and is targeted for proteasomal degradation.In our previour study,the OsOFP22-overexpressed transgenic rice plants demonstrated shortened internode of cu Lm,wider leave,increased chlorophyll content,rounder and thicker seeds and delayed flowering time.Overexpression of OsOFP22 confered more sensitive reaction to GA and reduced sensitivity to BL in the transgenic plants.To further explore the molecular mechanism of OsOFP22 in regulating plant growth and development,we construct Crispr/Cas9 vector for OsOFP genes cutting and genetic transformation.The phytohormone GA,ABA and auxin related genes were analyzed at their m RNA transcriptional level in theOsOFP22-overexpressed and OsOFP22-inhibited transgenic lines.The main results in this study are as follows:1.The construction of Crispr/Cas9 vector for OsOFP genes cutting.To decide the optimal guide RNA(g RNA)of OsOFP6,OsOFP19,OsOFP22,totally 12 g RNAs were designed with 4 g RNAs for one OFP gene.After annealing,the in vitro trancription was performed.The optimal g RNA for each OsOFP was confirmed by Cas9 digestion taking the ds DNA fragment(500-1000 bp)as the template which contains the target sequences of g RNAs.Then the Crispr/Cas9 vector for OsOFP6 and OsOFP22 or OsOFP6,OsOFP19 and OsOFP22 simultaneously cutting was constructed throught successively inserting the g RNA of the sigle gene into the vector.2.The identification and preliminary phenotype analysis of the Crispr/Cas9vector-transformed rice linesThe transgenic rice plants tranformed by Crispr/Cas9 vectors mentioned above were obtained through agrobacterium-mediated transformation method and were named BL5 and BL6 respectively.Both the resu Lts from PCR amplification for the specific sequence of the Crispr/Cas9 vectors and the m RNA expression level analysis of the target genes confirmed that the transcription level of OsOFP6 and OsOFP22 or OsOFP6,OsOFP19 and OsOFP22 were simultaneously inhibited in BL5 and BL6 lines.The transgenic T1 lines showed increased growth in plant height and lamina joint bending,indicating that OsOFP22 functions redundantly with OsOFP6 and OsOFP19 in repressing the BR response in the plant cell.3.OsOFP22 functions as a trancriptional repressor.The effector and reporter vectors were transfected into the Arabidopsis mesophyll protoplast.GUS reporter gene is driven by 35 S promoter linked with Lex A DNA binding or Gal4 DNA binding sequence.The effector vectors express VP16 transcrptional activator fused with Lex A DNA-binding domain(LD-VP16)or Gal4 DNA-binding domain alone(GD),or OsOFP22 protein fused with Gal4DNA-binding domain(OsOFP22-GD).Co-transformation of the reporter gene with the effector of OsOFP22-GD significantly reduced the GUS activity both in theabsence and presence of the LD-VP16 construct,indicating that OsOFP22 is a potent transcriptional repressor.4.The transcriptional level change of the phytohormone-associated genes is opposite between OsOFP22-overexpressed and OsOFP22-inhibited transgenic lines.The m RNA accumu Lation of genes involving in the phytohormone GA,ABA and auxin response was detected in OsOFP22-overexpressed trangenic lines,BL6 and the wildtype respectively.The results showed that the transcription of GAGA788,GA865 and GA888 was inhibited obvioursly in OsOFP22-overexpressed trangenic lines,whereas their transcription level was increased in the BL6 lines,compared to that in the wildtype.Contrarily,the m RNA accumu Lation of AUX769,ABA212 and ABA571 was elevated in OsOFP22-overexpressed trangenic lines,however,their m RNA accumulation was supressed in the BL6 lines,compared to that in the wildtype.These results indicated that OsOFP22 regulates multiple aspects of plant growth and development,which is likely achieved by adjusting the transcriptional levle of the phytohormone-associated genes.