Knockout And The Biological Functions Of OsGPRP Genes In Rice

Proline-and Glycine-rich protein genes?GPRPs?are widely present in plants.Many studies suggest that GPRPs may play an important role in plant growth and stress adaptation.However,no related studies on these genes in rice have been reported.In this study,the cis-acting elements in the promoters of rice OsGPRPs and their expresson patterns were analyzed by bioinformatic methods;OsGPRPs knockout and RNAi transgenic plants were obtained,and the genotypes of knockout progenies and the phenotype of homozygous transgenic mutants were identified.The main results are as follows:1.Eight abiotic stress-related elements,seven hormone-related elements and four other elements were identified in OsGPRP1,OsGPRP2,and OsGPRP3 with the quantities and types vary in each gene.OsGPRP2 and OsGPRP3 both contain abundant endosperm expression regulatory elements.2.Different expression levels of OsGPRPs were shown in different tissues of rice.The expression of OsGPRP1 was detected in all tissues,demenstrating a constitutive expression pattern,while OsGPRP2 specifically expressed in seeds 3days after pollination and the highest expression level of OsGPRP3 was recorded in seeds 10 days after flowering.3.Homozygous T₁ generation plants were obtained from a CRISPR/Cas9single gene editing system targeting OsGPRP3.Among them,13 strains were detected with a single base A insertion at the target site,18 strains with a single base T insertion,4 strains with a deletion up to 103bp,3 strains with a double base deletion and 1 strain with a single base deletion.All transgenic seedlings are under selfing.A multiplex CRISPR/Cas9 system targeting OsGPRP1,OsGPRP2,and OsGPRP3 was generated,and 60 T₀ transgenic positive plants were obtained.Among them,there were 4 transgenic plants with Os GPRP1,2,3 triple gene mutation,2 transgenic plants with OsGPRP1,3 double gene mutation,6 transgenic plants with OsGPRP2,3 double4.gene mutation and 2 transgenic plants with OsGPRP3 single gene mutation.5.Comparison on spike length and 1000-grain weight of homozygous OsGPRP3 single gene knockout transgenic lines and wild-type plants were performed.No significant difference of the panicle length and 1000-grain weight between transgenic and wild type plants were detected.Data on seed germination experiments showed that the germination rate of mutant was significantly reduced by 14%-40%compared with that of wild type,indicating that OsGPRP3 plays a role in plant growth and development.6.Drought stress was applied on homozygous OsGPRP3 single gene knockout transgenic lines and wild-type plants for 7 days.It was found that the degree of wilt in the transgenic plants was more serious than that in wild-type.The survival rate of the mutant was reduced by 36%-74%compared with that of the wild type after the recovery treatment,suggesting Os GPRP3 may play an important role in drought stress responses in rice.7.Fifty-five OsGPRP1-RNAi positive plants were obtained,and 54 of which were OsGPRP1 significantly down-regulated plants.23 OsGPRP2-RNAi positive plants were obtained with 7 OsGPRP2-significantly-down-regulated plants.15OsGPRP3-RNAi positive plants and 5 significantly down-regulated plants were obtained.These provide important experimental materials for further research.This study preliminarily indicates that OsGPRPs play important roles in the process of drought stress.Our work will provided elite genetic material for further study on the function of OsGPRPs in abiotic stress of rice with OsGPRP3 single gene knockout transgenic plants,two gene knockout transgenic plants,three gene knockout transgenic plants and RNAi transgenic plants we obtained.