The Map-based Cloning And Functional Analysis Of Maize Opaque-sk Gene

Maize(Zea mays)is one of the most important crops in the world and also an important organism for genetic and molecular biology studies.In maize endosperm,starch and storage proteins are the primary nutrient component and decide the yield and quality.The research on maize kernel development is a key step to improve the yield and quality of maize kernel.The phenotype of Maize opaque-sk(osk)is small kernel and opaque.Cytological analysis showed that the starch granules and protein bodies of osk mutants are smaller.Biochemical analysis showed that osk mutant endosperm displays decreased starch contents and increased protein and lipid contents.The total amino acid content of osk mutant endosperm is higher than the wild type and the free proline content of osk is four times higher than the wild type.The OSK gene was cloned by map-based cloning.Functional complementation and allelism test were performed to confirm the rightness of OSK gene.It encodes a basic helix-loop-helix(bHLH)transcription factor(TF).The phylogenetic tree showed OSK and its dicotylous homologous proteins can be clustered into two clades in evolution.Temporal and spatial expression analysis showed that in transcription level,only kernels had the expression of OSK and peaked at 9 DAP.In protein level,the OSK protein is only detectable in the endosperm of maize kernels and peakd at 18 DAP.Immunofluorescence assay showed that the OSK protein is mainly accumulated in the starchy endosperm(SE)and the embryo surrounding region(ESR).Subcellular localization and cellular fractions assay showed that OSK is mainly accumulated in nucleus and in cytoplasm there are also some signals.RNA-Seq analysis revealed 1,179 differentially expressed genes(DEGs)indicating osk mainly affects cellular carbohydrate and amino acid metabolic processes and stress response.Chromatin immunoprecipitation coupled to high-throughput sequencing(ChIP-Seq)analysis detected 9,080 DNA binding sites of OSK distributed over 4,019 genes.Overlay of the RNA-Seq and ChIP-Seq data revealed 182 potential OSK direct targets.Overall,OSK mainly regulates genes involved in carbohydrate metabolism and key regulators of protein and amino acid synthesis including 02(Opaque2),PBF(Prolamine-box binding factor)and cyPPDKs(Endosperm-specific cytoplasmic pyruvate orthophosphate dikinases).OSK also regulates the development of ESR by directly regulating genes specifically expressed in the ESR.Cold response genes and massive TFs are also direct targets of OSK.Through overlapping with 02 ChIP-Seq data,79 genes were found being regulated by both OSK and 02 and the regulation relationship of OSK and 02 for cyPPDK2 is antagonistic.This research indicated that the functions of OSK is mainly regulating carbon and nitrogen metabolism,ESR development and transcription of cold response genes.The direct targets of OSK revealed the mechanism of OSK regulation.At the same time,the studying of relationship of OSK and 02 complete the regulation networks of kernel development and it is useful to prove the yield and quality of maize kernel.