Identification,Gene Mapping And Cloning Of Maize White Kernel Mutants

Maize is an important food crop.It has an increasing share in feed processing and other industrial raw material processing.Therefore,the quality of corn is directly related to the nutritional health of humans and livestock.The color of corn kernels is varied and complex,and carotenoids are one of the substances that determine the color of maize kernels.Maize kernels are rich in carotenoids such as zeaxanthin,α-carotene,β-carotene,cryptoxanthin,and lutein,all of which are essential nutrients for the human body.Thus,identifying and analyzing maize color mutants,identifying key genes and cofactors of carotenoid biosynthetic pathways,and studying the molecular mechanisms involved in the generation of related mutants are of great significance for cultivating high-quality corn.In this study,the gene of white kernel mutant wk-15 obtained by EMS was cloned by map-based cloning method.At the same time,the mutant phenotype and physiological and biochemical indicators were analyzed.Additionally,the research results obtained are as follows:1.obtaining the white mutant wk-15 by the EMS method.(1)Phenotypic observation revealed that compared with the wild-type RP125,the mutant wk-15 showed white starch in the endosperm starch layer.(2)Scanning electron microscopy revealed that there were no significant changes in the number and structure of protein bodies and starch grains in the endosperm cells of the grain,and there was no difference in the detection of the gliadin content.(3)Measurement of mature grain size: The grain phenotypes of the wild type and the mutants were compared,there were significant differences in grain length and grain width(p < 0.01),and there was no significant difference in grain thickness and grain weight(p > 0.05).(4)The observation of grain at different developmental stages revealed that the color difference between the mutant wk-15 and the wild-type seeds appeared at 15 days after pollination,and then became significantly different with grain development.(5)Determination of grain carotenoid content: The content of zeaxanthin and carotene in the mutant wk-15 grain was significantly reduced(p < 0.01),and the content of zeaxanthin and carotene in the wild type was 30.8 μg/g and 5.6 respectively.mg/g;whereas only zeaxanthin was detected in the mutant at 3.5 μg/g,carotene was not detected.2.The B73×wk-15 F2 population was constructed.F1 hybrids had no yellow-white detachment,and white traits were isolated from the F2 population.It was verified by genetic analysis that the segregation ratio of yellow-white granules was 3:1,which proved that the mutant trait was implicated single gene control.3.Using B73 × wk-15 F2 generation segregation populations for gene mapping:construct BSA dominant recessive pool screening polymorphic markers for initial mapping and localize segments to maize chromosome 2;The In Del marker was newly developed as a fine localization marker,and the mutant gene was located between the ind-263 and ind-282 markers with a 200 kb segment size using 650 recessive individuals.By cloning and analyzing 10 candidate genes in the segment,the Zm00001d001909 gene was identified as a candidate gene.In the mutant wk-15,there is a single base mutation in the fifth exon of the gene,which is mutated from the "G" base to the "A" base,and the amino acid is changed from tryptophan(Trp)to a stop codon.The recessive pool and transcript amplification and sequencing verified that the site is accurate and real.Allelic detection of six similar white granulation mutants revealed an allelic mutant material,wk-308.4.The candidate gene Zm00001d001909,the full-length is 3412 bp,contains 9 exons,there are two transcripts,encoding 343 amino acids.The gene encodes an alternate oxidase(AOX),which is involved in electron transport in the mitochondria,and is tentatively named ZmAOX4.By phylogenetic tree analysis,the gene is highly conserved among species and has higher homology in rice and sorghum than Arabidopsis thaliana.The encoded protein is predicted to contain a Ferritin-like domain,and the tertiary structure is a dimer.5.The results of quantitative real-time PCR assay showed that ZmAOX4 gene has tissue-specific expression in the process of grain formation,and it showed relatively high expression in grain and seedling leaves,and the expression level in mutant wk-15 is obviously lower than that in wild type.q RT-PCR results of key genes in the carotenoid synthesis pathway: Compared with the wild type,four genes(PSY1,PDS,ZDS,crt RB1)showed significant differences in the expression of the mutants(p < 0.01 or p < 0.05),and their expression was affected;the other two genes,LCY-β and LCY-ε,were showed none difference expressed in the mutants(p > 0.05),the expression was not significantly affected.