Gene Mapping And Functional Analysis Of Maize Chloropyhll-deficient Mutant Apg3

Maize(Zea mays L.),as one of the five major grain crops(rice,wheat,maize,soybean,potato),is widely used in food,feed,chemical industry and other fields.At the same time,maize has the advantages of dioecious flowers,convenient artificial pollination,and a large number of seeds,and is often used as a model plant for genetic development research.Mutants are important materials for studying maize functional genes,and are of great significance for maize germplasm improvement and innovation.Maize mutants can usually be created by transposon insertion,physical mutagenesis,and chemical mutagenesis.Among chemical mutagenesis,EMS(ethyl methanesulfonate)mutagenesis is highly operable,simple and easy to implement,and usually produces G>A/C>T mutations,and the mutation sites are many and random Ly distributed.In recent years,the rapid development of high-throughput sequencing technology has made the rapid localization of maize EMS mutagenized mutants possible.In this paper,the gene mapping and preliminary functional analysis of a maize yellowing mutant(apg3)produced by EMS mutagenesis were carried out.The research progress achieved is as follows:1.Mutant phenotype identification and genetic analysis: The maize inbred line B73 apg3 mutant showed a yellow leaf phenotype after seed germination,and the seedlings gradually died after the three-leaf stage.Compared with normal plants,the content of photosynthesis-related pigments in mutant leaves was significantly decreased.Submicroscopic observation revealed that the grana lamellae of chloroplasts were lost in mutant mesophyll cells.Genetic analysis indicated that the mutant trait was controlled by a single gene recessively.2.Gene mapping: Gene mapping of the etiolated mutant was carried out by Mut Mup method,and it was found that the ZmAPG3(albino or pale green mutant)gene had a non-synonymous mutation of c.836G>A | p.G279 E.According to the structural and functional annotations,this gene encodes a polypeptide release factor,containing the typical peptide-releasing factor GGQ region,[ARH]-[STA]-x-G-x-G-G-Q-[HNGCSY]-[VI]-N-x(3)-[ST]-[AKG]-[IV],the mutation of p.G279 E occurs at the first relatively conserved G position in the above sequence.3.Allelic test: Allelic mutants of ZmAGP3 were obtained from the public EMS Mutant Bank(MEMD),which had a premature termination mutation in ZmAPG3,and the homozygous mutant seedlings exhibited an albino phenotype and died after the three-leaf stage.Gene allelic tests were performed on the yellow and albino mutants.The results showed that the two mutants were controlled by different alleles of the same gene.Therefore,it was proved that the mutation of ZmAPG3 could lead to the yellowing and albino phenotypes of maize.4.Analysis of gene function: The results of ZmAPG3 bioinformatics analysis showed that APG3 in maize had the highest similarity with the homologous genes in sorghum and southern Dips.ZmAPG3 was expressed in different tissues of maize,and the expression level was the highest in leaves.A plant transient expression vector was constructed for ZmAPG3 and transformed into maize leaf protoplasts.It was found that ZmAPG3 was localized in chloroplasts,and the p.G279 E mutation did not cause changes in the subcellular localization of the protein.At the same time,the expression changes of chloroplast development-related genes by ZmAPG3-p.G279 E mutation were analyzed by q PCR.In conclusion,this study carried out a more detailed phenotypic observation,gene mapping and preliminary functional analysis of a maize EMS-induced etiolation mutant.The results laid a theoretical foundation for the study of maize growth and development,photosystem morphogenesis and further genetic improvement.