The Positional Cloning And Functional Characterization Of Maize Mutant 1768

Maize is the highest-yielding crop in the world,and the issue of its yield and quality has attracted wide attention of breeders.Maize kernel is an important organ for nutrient storage and an important factor in determining its yield and quality.Maize kernel mutants are good materials for studying basic biological problems such as corn grain development and storage accumulation.The 1768 mutant is a kernel defective（dek）mutant controlled by a single recessive nuclear gene with the flat,opaque,starchy endosperm and ungerminable embryos.SDS-PAGE analysis indicated that the 27 kDaγ-zeins are reduced,but the 22 kDaα-zeins are increased slightly in 1768 mutant.Paraffin section showed that the embryonic development of the 1768 mutant was impaired,which may be an important reason why the mutant seeds could not germinate.In this study,after characterizing the F₂ population with 1090（372 wild,718 mutant）individuals,the 1768 gene was finely mapped in the physical interval of about 0.28 M on the short arm of maize chromosome 1.Within this 0.28 M interval,31 genes were identified.The genome sequencing and RNA-seq data analysis showed that the 1768mutant has an mutation from A to G in the 8564^th base of the Gene6 genome compared wtih the wild type.The A/G transition at the penultimate conserved nucleotide of the 21^st intron changes the splice site（GT-AG to GT-GG）.This mutation caused the deletion of 13 bps and a frame shift in the ORF of the 1768 mutant Gene6,resulting in a premature stop codon in the mRNA.Through the Protein BLAST alignment analysis,it was found that Gene6 encodes a Rrp44（Dis3）protein belonging to the RNR superfamily,which has both endonuclease activity and exonuclease activity.Phylogenetic analysis further demonstrated that this gene encodes the exonuclease Rrp44,one of the exosome complex subunits in eukaryotic cells.The Rrp44 protein is the tenth core subunit of the eukaryotic RNA processor exosome complex,which provides activity for exosome complex and is an essential protein in eukaryotes.In 1768,the A/G transition caused a premature stop codon in the mRNA resulted in the deletion of 83 amino acids in the protein sequence of 1768 mutant.These 83 amino acids contain the C-terminal S1 domain of Rrp44（Dis3）protein.Deletion of the S1 domain of Rrp44 protein results in embryonic dysplasia of the maize 1768 mutant,which affects the growth and development of maize mutant kernels.The research of Rrp44 in other species has been relatively mature,but there is no relevant research in Maize.This study intends to further explore the role of Rrp44 in maize grain development through the cloning and functional analysis of 1768 mutant gene.