Cloning Of Rice Grain Length 5 (GL5) And Exploration Of Variation On Its Promoter

Rice grain size is a key component of grain yield.Studying on grain size not only is meaningful to biology but also benefits rice breeding.Great progresses have been made in identifying grain size genes and uncovering the molecular mechanism in the recent years,cloning more grain size genes and analysis of molecular mechanism still display its great importance to rice production.In this research,we focused on the large grain size Minghui 63 EMS（ethyl methanesulfonate）mutant,named grain length 5（gl5）.Through gene cloning and nucleotide diversity analysis of GL5,we investigated the relationship between natural variation of GL5 promoter and the grain length,which may provide clues for exploring novel alleles resources.The main results are as follows:1.The gl5 mutant exhibited no significant plant architecture difference comparing to wild-types,while showed elongated grain length,reduced spikelet number,poor spikelet fertility and low yield.Through I₂-KI staining the mutant showed lower pollen fertility than wild type.2.We identified the candidate gene of GL5 using high-throughput sequencing and MutMap strategy,which locates on chromosome 5 and encodes OsMKP1,a homolog of Arabidopsis MKP1.A non-synonymous mutation in gl5 resulted in the changing of encoded amino acid.The positive genetic complementary plants showed similarity of grain length,fertility and spikelet number to wild-types.3.The knock-out plants based on CRISPR/Cas9 strategy exhibited large grain size,low fertility and reduced spikelet number per panicle in the background of Zhonghua 11,which is similar to the gl5 mutant.The transgenic overexpression plants showed short grain size and erect panicle.The RNAi lines showed correlation between the grain length and the suppressed RNA expression,indicating that the expression of GL5 was negatively correlated with grain length.4.We investigated the nucleotide variation of GL5 in germplasm population.The average nucleotide diversity of GL5 in indica,japonica and wild rice were much lower than that of the whole genome level and its flanking regions.Besides,theπof coding region was lower than its upstream promoter region.5.The promoters were classified into 11 haplotypes.These 11 haplotypes were cloned into plasmid to drive the luciferase expression in rice protoplasts.The relative luciferase activity that indicate the expression level of different promoters displayed significant differences.The varieties in germplasm population were classified into GS3 non-functional genotype and functional genotypes,and negative correlation was found between grain length and promoter driving ability in both groups,which indicating these different genotypes may affect the grain shape in a certain range.