Functional Analysis Of Seed Development Genes Emb23 And Emp6 In Maize

Corn is an important crop,the quality of seeds directly determines its grain yield and affects the subsequent production.Therefore,it is of great significance to study the molecular mechanism of seed development.In this thesis,the mutation of two genes Emb23 and Emp6 have seriously affected the maize seed development.The study of the function of these two genes will help to deepen the understanding of seed development regulation and provide theoretical support for breeding.During the co-evolution with host cells,many chloroplast and mitochondrial genes have been transferred to the nuclear genome,and their gene expression also requires nuclear-encoded proteins.There are 59 RPs(ribosomal proteins)in the plant chloroplast,which are encoded by the nuclear genome and the chloroplast genome.The ribosomal proteins and rRNAs composed the subunit of the ribosome,and then initiate the translation process.PORR(plant organelle RNA recognition)protein is a kind of RNA-binding protein found in recent years,distributed in chloroplast or mitochondria,mainly involved in the introns splicing process of the two organelles to help the genes expression.PORR proteins are relatively fewer in plants,but they are essential in RNA splicing.We obtained the emb23 mutant from the maize mutant library UniformMu.The selfed emb23 heterozygotes shows embryo lethal(emb,embryo defective)phenotype,and the wild type and emb kernels segregated in a 3:1 ratio,indicating that emb23 is a recessive mutant.Paraffin sections suggest that emb23 mutation seriously affected the development of embryos,but did not affect the endosperm.Emb23 gene was cloned by forward genetics approaches.It is 591 bp in length and had no intron,encodes a 196 amino acid protein.According to BLAST results,Emb23 encodes RPL10(Ribosome protein Large 10)protein.Gene expression pattern analysis showed that Emb23 was widely expressed in various tissues.By transient expression of EMB23-GFP fusion protein in tobacco cells,EMB23 protein was found localized in the chloroplast.This suggests EMB23 is involved in the chloroplast translation process as a chloroplast ribosomal protein.In the emb23 mutant,the chloroplast function is affected by the abnormal translation process,thus the development of the embryo is inhibited.Emp6 gene was reported in 2014,with a total length of 2291 bp,encoding a 408 amino acid PORR protein.Mutant kernels show emp(empty pericarp)phenotype with severely affected embryo and endosperm,and sharply decreased BETL cells.There are three allelic mutants of the Emp6 gene in the UniformMu mutant library,and the phenotype and genotype are closely linked as shown by crossing between different alleles.Subcellular localization revealed that EMP6 protein functions in mitochondria,but how the Emp6 gene affects seed development was not well studied.According to the RNA recognition property of the PORR protein,we hypothesize that Emp6 is involved in the post-transcriptional processing of precursor mRNAs.Therefore,RT-PCR was first used to detect the changes of mitochondrial intron splicing,and found that the splicing of nadl intron 1 and nad4 intron 3 and 4 was defective.The results of Blue Native Page and Western Blot showed that the assembly of complex I in mitochondrial electron transport chain was abnormal and the activity was absent.The expression of alternative oxidase AOXs were up-regulated at both transcriptional and protein level.These results indicat that lack of activity of complex I in emp6 mutant affects the electron transport of the NADH pathway,and thus activats the alternative respiration.Therefore,we can conclude that due to the lack of splicing in emp6 mutant,the assembly of complex I on the electron transport chain is abnormal,so that the oxidative phosphorylation cannot proceed normally,and the energy supply is insufficient,which ultimately affects the seed development.The above studies will help us to clarify the phenotype of two maize kernel mutants and the molecular mechanism of these two genes,which will allow us to further focus on the long-term goal of constructing a molecular network for maize development.