DA1 Related Genes Regulate Grain Size In Rice (Oryza Sativa L.)

Seed size is a major constituent of seed yield,which is largely determined by inherent genetic controls during the seed development.As an important agronomic trait,grain type is a quantitative trait controlled by multiple genes and susceptible to environmental impact,of which the genetic basis is complex.At present,a dozen of genes in rice had been identified to control grain length,grain width and grain weight,but the genetic and molecular mechanisms are still poorly understood.DA1 encodes a ubiquitin receptor,which sets final seed and organ size by restricting the period of cell proliferation.Seven other predicted proteins in Arabidopsis share extensive amino acid similarity with DA1 were named as DA1-related(DAR)proteins of which four were found in a tandem cluster on chromosome 5.DA1 and DARs were also found in rice and named as Os DA1,OsDAR2,OsDAR3 and OsDAR4 according to protein structural similarity with AtDA1 and AtDAR in Arabidopsis.Here,we investigated the functions of three OsDAR genes on seed size and overall growth of plant tissues.The OsDAR2,OsDAR3 and OsDAR4 overexpression transgenic lines showed no obvious phenotypes in germination,seedling growth,plant height,leaf length and number of tillers.However,the seeds of above overexpression lines showed enhanced grain length,grain width and increased TGW(thousand seed weight)in comparison with wild-type plants.In a sharp contrast to above overexpression lines,CRISPR/CAS9 knockout lines showed reduced grain length,grain width and TGW.These results demonstrated that OsDAR genes can increase seed size,TGW and grain yield per plant in rice.We further fused the VP16 transcriptional activation motif and the EAR transcriptional suppression motif with OsDAR2 and OsDAR3 respectively and obtained multiple transgenic lines.The phenotypes of VP16-DAR transgenic lines were similar with the overexpression lines,while the EAR-DAR transgenic lines were similar with the CRISPR/CAS9 knockout lines,indicating that OsDAR2 and OsDAR3 may function as transcription factors to regulate seeds development.Taking together,this research provided new clues to understand the regulating mechanisms of how DAR proteins control seeds size in rice.Moreover,the DAR genes may be widely utilized by breeders to alter the seed shape or increase crop yield in future.