Cloning And Functional Analysis Of ZmSMK118,ZmCYCB1-1 And ZmENOPH1 Controlling Kernel Development In Maize

As a multi-purpose crop for food,economics and forage,the yield of maize is very important for world food security and industrial development.Maize kernel is the biological basis for the formation of yield and quality,and its related traits are complex quantitative traits regulated by multiple genes.Therefore,cloning the genes related to grain development and analysis their molecular mechanisms are of great significance for the improvement of maize yield and quality.In this study,three genes related to grain size and quality were identified by EMS mutagenesis and other molecular biological methods.They were named Zm SMK118,Zm CYCB1-1 and Zm ENOPH1 respectively,and their functions were further verified by molecular biology experiments.The conclusions which may be drawn from this research are as follows:1.Cloning and functional verification of Zm SMK118Using EMS mutagenesis of maize inbred line Jing 92 pollen obtained a batch of mutants with research significance and breeding value,including a small-kernel mutant,smk118.Compared with the wild type,the mutants had significantly smaller kernels,and the 100-grain weight was about 40.35% of the control.The kernel development of smk118 was relatively delayed,but the offspring seeds can germinated normally.The mutant plants grown slowly at the seedling stage,and the plant height at the VT stage decreased by20.54% compared to the wild type.Genetic analysis demonstrated that smk118 was controlled by a single recessive gene.By Mut Map analysis,we located the candidate gene to a 33 kb region on chromosome1,including 4 candidate genes.Among them,Zm00001d028809 had G to A and C to T mutations at +10bp and +751bp from the start codon ATG,respectively,resulting in the mutation of the 4th amino acid from aspartic acid(Asp)to asparagine(Asn).The amino acid was mutated from leucine(Leu)to methionine(Met)at position 251.All of 4 candidate genes were used for knockout experiments and functional validation individually.The results showed that the seeds of Zm00001d028809-ko were significantly smaller than those from wild-type plants,which further proved that Zm00001d028809 is the target gene of smk118,named Zm SMK118.Zm SMK118 gene encodes a placenta-specific 8 protein(PLAC8).Homology analysis found that Zm SMK118 is homologous to the maize cell number regulator(CNR)gene family and the tomato fw2.2gene,and the latter were known to regulate grain/fruit size.This finding suggested that Zm SMK118 may be a new member of the maize CNR gene family.2.Cloning and functional analysis of Zm CYCB1-1The cell cycle is a critical process during plant embryo and seed development and its progression is regulated by cyclins.In this study,we cloned a maize B-type cyclin gene,Zm CYCB1-1,and demonstrated that overexpression of Zm CYCB1-1 significantly accelerated the growth rate of isolated embryos.In situ hybridization and toluidine blue staining indicated that Zm CYCB1-1 was highly expressed in the plumule of embryos,and the cells of the plumule were smaller,denser,and more regularly arranged in Zm CYCB1-1overexpression plants.Overexpression of Zm CYCB1-1 in maize increased ear length and kernel width,which resulted in enhanced kernel weight by increasing kernel width.Transcriptome analysis indicated that the overexpression of Zm CYCB1-1 affected several different metabolic pathways,including photosynthesis in embryos and leaves,and lipid metabolism in leaves.Conversely,knocking out Zm CYCB1-1 resulted in plants with slow growth.3.Cloning and functional analysis of Zm ENOPH1Enolase is a key enzyme in plant glycolysis,and its phosphorylation is essential for grain starch accumulation.In this study,we cloned a maize enolase-phosphatase 1 gene,Zm ENOPH1.Bioinformatics analysis shows its evolutionary conservation.Study confirmed that loss of Zm ENOPH1 led to stunted growth,uniform leaf yellowing,reduced grain starch content and decreased grain weight.Transcriptome results showed that the deletion of Zm ENOPH1 affected the pathways of glycolytic metabolism,starch and sucrose metabolism in grains.The untargeted metabolome showed that deletion of Zm ENOPH1 resulted in decreased synthesis of flavonoids and flavonol metabolites in grains.