OsGPRP3 Affects The Growth And Development Of Grains In Rice

Glycine and proline-rich protein(GPRP)are widely distributed in plants,which can function in plant growth and development.However,Whether OsGPRPs are involved in grain size regulation has not been reported.In this study,we focus on the function analysis of OsGPRP3 in rice grain size,grain weight and seed germination via bioinformatics,CRISPR/Cas9 and cytology.Our results will lay a foundation for further studying on the biological function of OsGPRP3 and other plant GPRP family genes.The main results are as follows:1.The basic characteristics of OsGPRP3.The full-length CDS(513 bp)and genomic DNA(1831 bp)sequence of OsGPRP3(located on chromosome 5)were obtained using bioinformatics.OsGPRP3 is composed of two exons and one intron,encoding a total of 170 amino acids.The isoelectric point(pI)of the OsGPRP3 is 7.53 and its molecular weight(Mw)is 16.8 kDa.2.The expression pattern of OsGPRP3.qRT-PCR was performed to investigate the expression pattern of OsGPRP3 in different tissues of ZH11,and OsGPRP3 was found to be ubiquitously expressed.Among vegetative organs,OsGPRP3 was highly expressed in flag leave,stems,roots and the seeds at 10 days after pollination.To gain insight into the spatiotemporal expression pattern of OsGPRP3,promoter analysis was performed in transient assays using a glucuronidase(GUS)reporter gene.The results of GUS staining analysis showed that OsGPRP3 was highly expressed in flag leave,stems,roots,glumes of the day before flowering and the seeds at 10 days after pollination.In situ hybridization results showed that OsGPRP3 was expressed at significantly higher levels in flag leave,stems,roots and the seeds at 10 days after pollination.3.Creation of knockout mutants and measurements of their grain traitsThree kinds of knockout homozygous mutants(+A,-C,-102bp)were obtained by CRISPR/Cas9.Amino acid sequence analysis revealed that the conserved protein domains of the protein changed to varying degrees.Investigated the influence of OsGPRP3 on yield-related traits and first found that the grain size showed obvious differences among the ZH11 and osgprp3 lines.Compared with ZH11,the grain width and grain thickness substantially decreased in osgprp3.The 1,000-grain weight decreased substantially in the osgprp3 lines.4.SEM analysis of glume shell and grain storage material detection of OsGPRP3knockout mutant.The cells in the outer epidermis of glumes were investigated by SEM.The spikelet cell of osgprp3 was thinner than that of ZH11.The measurement datas indicated that OsGPRP3 may affect cell width along the grain-width direction to regulate seeds development in rice.The results of grain storage material detection showed that the total storage protein and lipid contents in the osgprp3 were significantly declined.We speculated that OsGPRP3 gene may be involved in the regulation of the rice grain filling process,affecting the weight and quality of rice.5.Knockout OsGPRP3 causing seed low germination ratesThe results of seed germination experiments in water showed that the seed germination rate of osgprp3 was significantly lower than that of ZH11,suggesting that OsGPRP3 gene may also affect the germination of rice seeds,but further experiments are needed to confirm this result.