Gene Cloning Of Maize Kernel Mutants T902-1 And Yys223 And Binding Factor Mining Of The Mn1 Promoter

Maize is a key resource for food,feed and industrial raw material production,and improving its yield and quality is an important guarantee to ensure national food security and stable economic development.The kernel is the main organ of yield,so it is important to explore the key genes affecting the development and filling of the kernel and to analyze the molecular mechanism of their action,which is an important theoretical guidance to improve the yield of maize.In this study,two kernel natural mutants found in the field were used as materials,and their candidate genes were obtained by phenotypic analysis and map cloning,and the core promoter of the candidate gene Mn1 was screened and identified as an extremely binding protein,and the main findings were as follows:1.Mapping cloning of grain mutant t902-1The kernel length,width,thickness and 100 kernel weight of the t902-1 mutant were significantly lower than wild type,The kernel length,width,thickness and 100 kernel weight of the t902-1 mutant were significantly lower than those of the wild type.The difference between the t902-1 mutant and the wild type appeared on the 8th day after pollination,and gradually increased with the development process.Cytological analysis showed that,compared with wild-type kernel,mutant kernel had delayed embryo and endosperm development,and less endosperm basal metastasis layer and cell wall hyperplasia.Genetic analysis revealed that the t902-1 phenotype is a recessive trait controlled by a single gene.Using map based cloning,the target gene was located between the S04164 and C18737 markers on chromosome 2,with a physical distance of 2.2 Mb.Sequence analysis found that there was a specific missense mutation site in the mn1 gene within the interval that led to the amino acid change from Glu to Asp.Allelic test results showed that t902-1 was a new mn1 allelic mutant.As a specific cell wall invertase in the basal metastasis layer of endosperm,Mn1 regulates sucrose release from phloem,catalyzed irreversible hydrolysis of sucrose to glucose and fructose in the extracellular body,and controlled sucrose absorption rate.However,the trans-acting factors that regulate the expression level of Mn1 are still unclear.Using the genome of maize inbred line B73 as a template,a sequence of 2000 bp upstream of the Mn1 start codon ATG was cloned.Combined with informatics cis element analysis and gene gun in vivo activity detection,it was determined that the sequence between-1000 and-501 bp upstream of ATG was the core promoter of Mn1.Yeast single hybridization and Dual Luc experiments have preliminarily determined that Zm MRP-1 and O2 are important trans acting factors that regulate the expression level of Mn1.2.Mapping cloning of the kernel mutant yys223The kernel length,width,thickness and 100 kernel weight of the yys223 mutant were significantly reduced.Genetic analysis indicates that the yys223 mutant phenotype is controlled by a single recessive nuclear gene.The gene was mapped between markers W73635 and W73716 on chromosome 8 of maize with a physical distance of 1.95 Mb.There is a gene Emp7 encoding PPR protein in this region,which is the key gene for the assembly of mitochondrial oxidative phosphorylation complex.Sequence analysis revealed the insertion of a single base G in the exon of the emp7 gene in yys223,resulting in a frameshift mutation and premature termination of protein translation.The allelic test results indicate that yys223 is an allelic mutant of emp7.