Cloning And Functional Analysis Of Panicle Traits Gene OsGRF4 In Rice

Rice is one of the most important staple crops worldwide.Panicle traits such as grain shape,panicle size,seed germination/dormancy and seed shattering,which controlled by quantitative trait loci(QTL)are complex yield determinants,play important roles in grain yield and grain quality in rice.Although numbers of genes controlling panicle traits have been cloned over the past decades,the molecular mechanisms underlying them are still unclear.Therefore,identification and function analysis of QTLs that regulate panicle traits enhance our understanding of rice panicle development.In this study,the cloning,functional analysis and application in breeding of a novel growth regulating factor OsGRF4 is reported.OsGRF4 positively regulates grain shape,panicle length and negatively regulates seed shattering.Our findings provide a novel insight into the molecular regulatory mechanisms controlling panicle traits,and also generate gene resources and technical support for high yield breeding of rice.The results are summarized as follows:1.By fine mapping,OsGRF4 was located in an interval of ~33.2 kb on rice chromosome 2,three putative gene loci,LOC_Os02g47280,LOC_Os02g47290 and LOC_Os02g47300,exist in this candidate region.LOC_Os02g 47280 encodes a putative growth-regulating factor OsGRF4,which belongs to the GRF protein family.Overexpression and RNA interference(RNAi)transgenic plants showed that LOC_Os02g 47280 was the candidate gene.OsGRF4 was preferentially expressed in developing panicles,especially in panicles of 7 cm in length.The transcript was much more abundant in NIL-OsGRF4 than in NIL-Osgrf4 in the young panicles.2.There were six nucleotide differences in the coding sequences of OsGRF4 between R1126 and CDL,resulting in the substitution of 3 amino acids.It was reported that OsGRF4 is downregulated by miRNA396 during grain development in rice,the SNP3(AA-TC)occurs at the OsmiRNA396 target site of OsGRF4,and this unique mutation of CDL was not found within the 25 rice accessions and the database involved over a thousand varieties.The abundant polymorphisms were also detected in promoter sequence.To determine the unique SNP3 mutation or promoter regions of OsGRF4 responsible for the phenotypic variation,fusion expression vector were constructed.Collectively,results from the sequencing,expression pattern and genetic transformation suggested that the specific mutation at the OsmiRNA396 target site of OsGRF4 coding sequence may enhance the expression of OsGRF4,and results in increased grain size,panicle length and improved seed shattering.3.By sub-cellular localization analyzing,the OsGRF4 protein co-localized to the nucleus with Ghd7.The observation of anatomical structure indicated that OsGRF4 gene improved seed shattering via differential abscission zone formation.OsGRF4 positively regulated grain size mainly by increasing cell length partially and cell number,leading to enhanced longitudinal growth in the grain.Four kinds of cytokinins showed significant differences between the two OsGRF4 alleles,the high levels of cytokinin regulated by cytokinin-related genes might result in larger grain,longer panicle and lower seed shattering.The functional marker of OsGRF4 was designed base on SNP3 mutation.4.We compared the transcriptome profiling of the 7 cm young panicle between NIL-OsGRF4 and NIL-Osgrf4 using RNAs sequencing,a total of 80 significant differentially expressed genes(DEGs)were identified.24 DEGs were selected for real-time PCR,result revealed the same expression pattern of all the selected genes in real-time PCR analysis as obtained from transcriptome sequencing data.Hierarchical Cluster analysis showed that OsGRF4 tightly clustered together with the up-regulated DEG LOC_Os02g47320.Transcription factor(TF)Osmyb1 also exhibited differential regulation between NILs,maybe is a key regulator for panicle trait.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis suggested that the DEGs were mainly involved in somatic embryogenesis,chitinase activity,amino sugar and nucleotide sugar metabolism.By co-localization of DEGs with previous identified QTL,a total of 54 DEGs were co-localized within 50 QTL responsible for grain shape,panicle length,seed shattering,seed set percent,seed germination/dormancy and cold/drought tolerance.These results provide new potential candidates genes for further understanding the molecular mechanisms involved in rice panicle traits regulation.5.Hybrid rice restorer line R257 carrying OsGRF4 gene was developed,and R257 was crossed with an indica two-line male sterile shuang8 S.From the results of variety comparative test in 4 different ecological regions(Hainan,Huaihua,Longshan and Cili),the hybrid combination from R257 and shuang8 S significantly increased its thousand seed weight,and produced the highest yield in Longsan,with a yield of 742.5 kg per mu,ranked No.1 in all combinations and all test sites.The yield of shuang8S/R257 was 14.7% and 5.6% higher than that of control Y Liangyou 1 and Fengliangyou 4,respectively,which indicated that OsGRF4 locus have high yield potential.