Functional Research Of Grain Size Gene OsRLG1 In Rice(Oryza Sativa L.)

The continuous increase of the global population and the stagnation of grain yield are the major problem of world at present.Rice is one of the important food crops,how to improve rice yield has always been the focus project of breeders.To raise the rice yield,it is necessary to improve the four elements of rice yield.Among them,rice grain weight is one of the four elements,and there is a positive correlation between rice grain weight and grain size.Therefore,discovering and utilization of rice grain size regulatory genes is of great significance for the cultivation of high-yielding rice varieties.In this study,OsRLG1,an important new gene regulating rice grain size,was cloned.The regulation mechanism of grain size for OsRLG1 was systematically analyzed,and the usable genetic variation analyses of OsRLG1 in rice germplasm resources were conducted.The specific research results are as follows:1.Through the analysis of tissue expression profile,it was found that OsRLG1 was highly expressed at the early stage of rice young panicle differentiation,and its expression level was gradually down-regulated during the growth and development of young panicle.Subcellular localization experiments discovered that OsRLG1 was localized in the nucleus.2.The gene editing and gene silencing transgenic materials of OsRLG1 with rice Nipponbare as the background were created and comparing to the wild-type Nipponbare for grain shape,it was found that the grain length and 1000-grain weight of the gene editing and gene silencing（RNAi）materials were significantly larger than those of the wild-type material,while the grain width and plant height showed no significant difference.The analysis showed that the expression level of OsRLG1 in RNAi materials was negatively correlated with the grain length of rice,R²=0.9139.When the full-length CDS of Nipponbare OsRLG1 was expressed in the mutant materails,and the complementary plants recovered the phenotype of rice grain size.All results confirmed that OsRLG1 negatively regulates rice grain size.3.Through scanning electron microscopy,it was revealed that the increased glume cell length of the gene editing materials of OsRLG1 was the direct cause of the increased grain length.4.Two genes（OsKin L and OsLNG）interacting with OsRLG1 were identified by yeast two-hybrid assay and Bi FC experiment.The functions of OsKin L and OsLNG have not been reported.It was showed that OsKin L and OsLNG were localized in the nucleus like OsRLG1.The transcriptional expression levels of OsKin L and OsLNG in gene editing mutants of OsRLG1 were significantly different from those of the wild type,which indicated that they could be involved in the regulation of rice grain size.5.According to the functional domain,OsRLG1 was truncated to form different truncated fragments.It was found that the complete DUF domain was the necessary region for the interaction between OsRLG1 and its interacting proteins.N-terminal and C-terminal amino acids outside the DUF domain of OsRLG1 may influence the strength of the interaction between the DUF domain and the interacting protein.6.The T-DNA inserted mutant rice materials of gene OsLNG were identified,and the functional complementary transgenic material for the mutant were developed.Through comparing the grain shape related traits of the homozygous mutant lng,its isolated wild-type and functional complementary materials,it was found that the grain length and 1000-grain weight of the mutant lng were significantly higher than those of the wild-type materials,the phenotype of functional complementary materials was recovered.These results suggested that OsLNG negatively regulated grain length and 1000-grain weight in rice.Using the same method,it was confirmed that OsKin L also negatively regulated grain length and 1000-grain weight in rice.Compared with the wild type,OsRLG1 was significantly down-regulated in mutant kinl.Compared with the wild type,the transcriptional expression level of OsRLG1did not change significantly in mutant lng.7.Three haplotypes of OsRLG1 named Hap＿1,Hap＿2 and Hap＿3 were identified by sequence alignments of 254 rice germplasm resources.The rice grain length of haplotype Hap＿2 was significantly larger than those of haplotype Hap＿1 and Hap＿3.There was no significant difference at rice grain length between haplotype Hap＿1 and Hap＿3.The distributions of the three haplotypes were clearly different in different rice resources.8.The OsRLG1 coding sequence of Nipponbare（Hap＿1）was transformed into MH63（Hap＿2）,the grain length and 1000-grain weight of MH63^NPBlines were significantly smaller than those of MH63 lines,and there was no significant difference at grain width and plant height.The results showed that haplotype Hap＿1 and Hap＿2 were alleles of OsRLG1,and Hap＿1 was the dominant allele.