Map-based Cloning And Functional Analysis Of OsUBP15,a Grain Shape Regulator In Rice

Rice?Oryza sativa L.?,one of the most important cereal crops,provides calorific intake for more than half population of the world.Grain size and shape are critical factors that simultaneously affect grain yield and grain quality of rice,which consisting of grain width,grain length,and grain thickness,and grain length-to-width ratio.The comprehensive understanding of the molecular mechanism of rice grain shape regulation is significantly important for breeding for high yield and quality cultivars.Although some genes that control rice grain shape have been cloned,the precise regulation mechanism has not yet been elucidated,and more genes need to be discovered.In this study,we identified a partially dominant mutant with wider and larger grain of rice,named large grain 1-D?lg1-D?.And the cloning and functional analysis of the O_SUBP15 gene was performed.The main results are as follows:1.Compared with the wild type,the grain width,grain length and grain thickness of the mutant lg1-D were increased by 30.8%,13.23%and 13.8%,respectively,and the 1000-grain weight increased by more than 30%.The plant and the panicle of lg1-D became short and erect.Although the tiller number per plant and the grain number per panicle were decreased,the yield per plant of lg1-D did not change significantly.The filling rate of mutant lg1-D was significantly higher than that of wild type from the ninth day after flowering,and dry matter accumulation was significantly higher than that of wild type from 12 days after flowering.2.Observation of the cross-section of the middle part of hull before flowering showed that the number of parenchyma cells of lg1-D was increased by 18.4%,and the cell length was increased by 5.1%,revealing that the increase of grain width in lg1-D resulted mainly not from the change of the cell size,but from the change of cell number.Scanning electron microscopic observation of spikelet hull showed that the epidermis cells of lemma in lg1-D are shorter than that of the WT,indicating that the increase of grain length of lg1-D was resulted from the increase of cell number of spikelet hull in longitudinal direction.Furthermore,the expression level of some cell cycle related genes were increased in the young panicles of the lg1-D mutant.The results demonstrated that lg1-D increases grain size likely by influencing the cell proliferation in both latitudinal and longitudinal direction of the spikelet hull.3 Genetic analyses showed that the lg1-D mutation behaved in a partially dominant manner.The mutated locus was delimited to an 89 kb DNA region on the short arm of Chromosome 2,using the F₂mapping population derived from the cross lg1-D and an indica variety Xieqingzao B.There were six open reading frames?ORFs?located in this region.Sequencing analysis revealed a single-nucleotide substitution in the coding region of ORF2?LOC₀2g14730?,which resulted in a premature protein in the lg1-D mutant.Transgenic complementation and interference verified that the mutation of ORF2 caused the mutant phenotype,and demonstrated that ORF2 positively regulates rice grain width,and lg1-D is a gain-of-function mutation.4.The LOC₀2g14730 encodes an ubiquitin-specific protease?OsUBP15?.Quantative RT-PCR and GUS staining demonstrated that OsUBP15 showed a constitutive expression pattern and was expressed in roots,stems,leaves,leaf sheaths,young panicles,and developing kernels.The subcellular localization analysis of OsUBP15 protein with the gOsUBP15-GFP transgenic lines revealed that OsUBP15 was located in both the nucleus and the cytoplasm.5.Deubiquitination assays showed that both OsUBP15 and osubp15 had de-ubiquitination activity in vitro.Combined with the results of the transgenic analysis above,we suggested that the loss of the C-terminal in Osubp15 did not disrupt its de-ubiquitination enzyme activity,and that the osubp15 protein was functional in vivo.The transient expression in tobacco and rice protoplasts showed that the accumulation of osubp15 was significantly higher than that of OsUBP15,indicating that he stability of osubp15 protein was promoted.6.The results of Yeast two-hybrid and Pull-down demonstrated that OsUBP15 can interact with OsDA1,a homologous protein of arabidopsis DA1,and OsDA1 can regulate the degradation of OsUBP15protein,which is consistent with the report in arabidopsis.The mutant protein osubp15 also has the ability to interact with OsDA1,but it was found in yeast two-hybrid experiments that the interaction between osubp15 and OsDA1 was weaker than that between the wild-type OsUBP15 protein and OsDA1,which indicated that the deletion of 160 amino acid residues at the C-terminal of the Osubp15 protein interfered its interaction with OsDA1.7.The loss function mutants of GW2 and OsUBP15 and double loss function mutants were constructed by CRISPR/CAS9.Genetic analysis of OsUBP15/LG1 and GW2 indicated that the two genes are not completely independent in regulation of grain width?Fig.7?.It is possible that GW2 and OsUBP15share downstream substrates during ubiquitination and deubiquitination,in addition to their specific roles.