Cloning And Functional Analysis Of The Dek559-2 In Regulating Maize Seed Development

Maize(Zea mays L.)is the important food and feed crop.Seed development is an important factor to determine its yield and quality.Therefore,the research on genetic regulation mechanism of maize seed is of great significance.In recent years,some mutants of seed development had been reported subsequently,the study of these mutants assisted us in making clear the genetic regulatory networks of maize seed development.Due to the development of corn seed is a complex process of gene regulation,their genetic regulatory mechanism is not clear.Screening new corn seed development mutants,cloning related genes and performing functional will promote us to discovery the key gene of corn seed development and analysis the genetic regulatory networks.It will provide the insights for the genetic improvement of corn yield and quality.Used EMS mutagenesis screening to obtain multiple mutants of seed development,the mutant of seed development,dek559-2 was screened,whose seed performed significantly smaller,withered,embryo and endosperm dysplasia.Genetic analysis showed that these seed developmental defects were controlled by a single recessive nuclear gene.Used molecular marker positioning,the target gene Dek559-2 was identified when combined with the sequencing comparison and expression analysis of candidate genes.The functional preliminary study was carried out,which will shed the light on the subsequent analysis of the molecular mechanism of seed developmental defects caused by Dek559-2 mutated.1.Isolation and Characterization of dek559-2 mutants.Morphological analysis showed that the seeds of the mutant dek559-2 were significantly smaller than wild-type(WT)and the hundred-kernel weight of dek559-2 was only about 12%of WT.In addition,the seed germination experiment revealed that the mature dek559-2 seeds could not germinate.Since seed germination was influenced by a variety of factors such as seed maturity,the pollination of 24-day-old immature embryos in mutants and WT were respectively stripped to further clarify the effect of Dek559-2 mutation on embryo development,showed that immature embryo in dek559-2 was significantly smaller than WT.Germination experiments were carried out on 1/2 MS culture medium,and the germination force of dek559-2 was significantly lower than WT,only about 35%of WT.The observation of paraffin section showed that the seed development of the mutant dek559-2 was significantly delayed and the number of starch granules decreased significantly when compared with WT.These results showed that the number of starch granules in dek559-2 endosperm was significantly reduced,and there were many holes on the surface.Therefore,dek559-2 mutation seriously affected the development of maize embryo and endosperm.2.Genetic analysis of dek559-2 mutants.Statistical analysis and chi-square test of normal and defective kernels in the ear of W64/dek559-2F2 group,it was found that the genetic separation ratio was consistent with the Mendelian first law of separation of 3:1,indicating that the seed developmental defect phenotype of dek559-2 is a recessive trait controlled by a single nuclear gene.3.Map-based cloning of Dek559-2.The DNA was extracted from the mutant grains in the ear of W64/dek559-2 F2 group,and the Dek559-2 was in the physical interval of 230 Kb on chromosome 3 using Indel marker.There are four predictive genes in the region,named Gene1-4.Quantitative Real Time PCR analysis showed there had no significant difference in expression quantities among these four genes in dek559-2 and WT.DNA and cDNA sequence comparison found that the base sequence of Gene2-4 in dek559-2 was exactly same as WT.But the second base of Genel's second intron had a T-to-C mutation,which caused the introns could not be spliced and premature translation termination.Further transgenic functional verification of this gene was currently being to do.4.Expression pattern and subcellular localization of Dek559-2.Expression analysis showed that Dek559-2 was expressed in maize seed coat,embryo and endosperm.Subcellular localization results indicate that the Dek559-2protein was localized to the cytoplasm and nucleus.5.Dek559-2 mutation breaks the oxidation-reduction balance of cells and affects the development of seeds.Dek559-2 encodes an unknown protein,belonging to Sufl superfamily,which contains multiple cupredoxin structural domains and it is most likely involved in the the oxidation-reduction regulation of cells,in turn affects ROS levels.The two major components of ROS(H2O2 and O2-)were tested by DAB and NBT staining respectively.The results showed that the contents of H2O2 and O2-in dek559-2 seeds were significantly increased.Further quantitative analysis of H2O2 by hydrogen peroxide detection kit showed that the concentration of H2O2 in dek559-2 seeds was about 2.7 times that of WT.The increase of O2-content was usually accompanied by increased activities of NAD(P)H dehydrogenase.The activities of NAD(P)H dehydrogenase in dek559-2 seed was found to be upregulated 4 times than WT by the NADPH oxidase kit.The above results indicated that the Dek559-2 mutation did result in a significant increase of ROS content in dek559-2 seeds.A large increased ROS can cause cell damage and even induce PCD.Combined with morphological analysis,we hypothesized that the aggravation of PCD induced by increased ROS may be an important reason that resulted in seed development defects of dek559-2,and it was currently being confirmed by TUNNEL kit.