Functional Analysis Of OsGIF1 Gene In Rice

Rice as the main food crop in China,to ensure its production is important for food security strategy.The yield components of rice were not only affected by grain size,1000-grain weight and seed setting rate,but also related to plant height,leaf type,panicle number and grain number per spike.Therefore,it is very important to clone and analyze the rice yield-related genes.Our previous study found.OsGIF1 and OsGRF4 interact with each other to form a transcription complex to act on the downstream target genes,and then regulate the rice seed,we previously showed that upregulation of OsGIF1 expression improves rice grain size.However,whether OsGIF1 is involved in other developmental processes and other agronomic traits remains uncertain.Therefore,in order to explore the function of OSGIF1,this study through a series of means of research(e.g.knockout,overexpression,expression pattern analysis and subcellular localization)for further analysis of the function of the gene.The main results were as follows:1.The knockout analysis of OsGIF1 was performed using CRISPR/CAS9 technique.The plasmids were then transformed into the wild-type(WT)variety of Nipponbare(Nipp),and approximately 30 transgenic plants were obtained for each plasmid.Sequencing of PCR-amplified OsGIFl genomic DNA from transgenic plants showed five types of homozygous mutations within the target sites:one in target site 1(an A insertion)and four in target site 2(3-base deletions,18-base deletions,10-base deletions,and an T insertion),indicating that the target gene was edited successfully.The phenotypic comparison of mutants and wild type showed that OsGIF1 knockout plants showed different degrees of variation in plant height,which were slightly dwarfed,moderate dwarfed and extremely dwarfed.This is a plant and dwarfing OSGIF1 gene knockout due to the degree of change related protein sequences,such as A,T OsGIF1 base insertion can cause premature termination of the encoded protein,corresponding extreme dwarf plants can also occur.At the same time we found no off-target.Therefore,the phenotype was directly caused by OsGIF1 mutation,and the phenotypic diversity of the different mutation types might have resulted from varying degrees loss of OsGIFl function.1.OsGIF1 KO has pleiotropic effects on rice development.We selected three homozygous mutant strains(T408-14,T408-6 and T409-17)from the knockout plants of OsGIF1,and analyzed the function of OsGIF1 by comparison with wild type plants.These results demonstrated that functional loss of OsGIF1 led to reduced plant height of rice by shortening the internode length of stems.Leaf lengths and leaf width of all the mutant plants were significantly reduced compared with that of the WT.The leaf rolling indices of plants with the most severe mutations,namely T408-6 and T409-17,were sharply increased,with almost all leaves rolled compared with those of the WT.These results indicated that OsGIF1 affected rice leaf development by regulating both leaf size and leaf rolling.In addition,the 1000-grain weight,grain length and grain width of OsGIFl knockout mutant were significantly decreased,indicating that OsGIF1 affected the growth and development of rice grains.Moreover,the OsGIF1 knockout mutant showed a significant decrease in floral organ developmental abnormality and seed setting rate,especially T409-17,which was almost completely sterile,suggesting an important role of OsGIF1 in the determination of rice floral organs.3.Overexpression of OsGIF1 increased the size of multiple rice organs.2x35S was used as promoter to over express OsGIF1.overexpression of OsGIF1 in these plants also very significantly increased the grain size and weight by synchronously increasing the grain length and width.Additionally,overexpression of OsGIF1 apparently affected leaf development,because all these plants exhibited very significantly increased leaf length and width.These results confirmed that overexpression of OsGIF1 increased the size of multiple rice organs,indicating a positive role of OsGIF1 in regulating rice organ size.4.OsGIF1 controls rice organ size possibly by regulating cell expansion.The Cytological comparative observation of stem and leaf of wild type and OsGIF1 knockout was carried out by paraffin section.The number of cells per unit area of Stem internodes Slanting,crosscutting and leaf crosscutting in mutant plants was significantly increased,and the cell size was significantly reduced.At the same time,the glume cells size of Overexpression individual plant significantly increased by the observation of the glume of wild type,overexpressed and knocked out plants by scanning electron microscopy,and the glume cells size of Knock out individual plant was significantly decreased.these results suggest that OsGIF1 enhances the size of multiple important rice organs predominantly by promoting cell enlargement.5.We also observed other morphological abnormalities except for cell size alternation in these plants,the layers of sclerenchyma cells of the stem vascular bundle and the number and size of the stem vascular bundle were apparently decreased in the KO plants.,the outer vascular bundle appears incomplete development in the most serious KO plants.Notably,the number of bulliform cells in the leaves of the serious KO plants was increased,which abolished the balance of the Ad-Ab patterning and consequently led to rolled leaves.Finally,scanning electron microscopy showed that OsGIF1 overexpression and knockout of plant leaves and stems of the outer surface of the arrangement of siliceous cells,stomatal guard cells morphology,number and arrangement of patterns and the wild type are significantly different.These results suggest that OsGIF1 might also affect other cellular processes during rice organ or tissue development.6.Expression Profile of OsGIF1.The expression pattern of OsGIFl was analyzed by GUS reporter gene system and qRT-PCR,The results suggested OsGIF1 Expression was relatively weak in the root and mature glume,but was strong in the internode,node,the developing spikelet,and especially in the developing anther.Overall,the expression pattern of OsGIFl is consistent with its function.7.The subcellular localization of OsGIF1.In order to investigate the subcellular localization of OsGIF1,we constructed an OsGIFl-YFP fusion construct with its expression.Transient expression in the rice protoplast cells showed that OsGIFI-YFP localized preferentially in the nucleus and was weakly expressed in the cytoplasm.This result is consistent with the idea that GIF proteins function as transcriptional co-activators by forming complexes with GRF transcription factors.