DAP-seq Analysis of Transcription Factors Associated with Maize Endosperm Development

Transcription factors (TFs) play an important role in maize endosperm development regulation. The temporal RNA-seq and co-expression network analysis of maize endosperm reveals a hiararchical regulatory network architechture modulating the endosperm development. In the network, NKD1 and NKD2 are central regulators, and GBF1, HSFTF10, NACTF49 and HB115 are secondary regulators downstream of NKD1 and 2. To further test the relationship between these TFs, a DNA affinity purification and sequencing (DAP-seq) assay was performed. The result shows that the assay on NKD1 and NKD2 called 1,951 and 56,855 peaks, and 93 and 1,692 peaks are located within 3 kb distance to transcription starting site (TSS), respectively. And assays on GBF1, HSFTF10, NACTF49 and HB115 called 15,543; 38,147; 7,388 and 936 peaks, respectively. Overall design: The open reading frames (ORFs) of genes of interest are cloned into Gateway entry vector, which are then recombined into the pIX-HALO vector using LR clonase II. Proteins are in vitro expressed using the TnT® SP6 High-Yield Wheat Germ Protein Expression System and immoblized on Magne HaloTag beads. The immobleized proteins then bind to maize B73 genomic DNA library, followed by washing and elution. The eluted DNA is PCR amplified and barcoded for sequencing. The barcoded DNA was sequenced on a NextSeq500 with 75 bp SE reads for GBF1, HSFTF10, NACTF49 and HB115 and on Novaseq with 150 bp PE for NKD1 and 2.