Research Projects

small RNA plays a crucial role in reinforcing transposable elements (TEs) silencing during epigenetic reprogramming in pollen development. These small RNAs are classified into miRNA and siRNA, with miRNAs controlling endogenous genes and siRNAs producing from repeats and TEs through the RNA-directed DNA methylation pathway. The mechanism of siRNA-mediated silencing of TEs in pollen development is not well understood, and it is unknown whether siRNAs are produced during pollen development or inherited from the sporophyte through meiosis. This project aims to identify the importance of siRNAs in transposon silencing and genome integrity during pollen development. The Pol IV/OsNRPD1 gene will be specifically knocked out in rice using a homologous recombination-mediated gene targeting approach. The heterozygous OsNRPD1 line will be used to sort out WT and osnrpd1 knock-out pollens. The study will use the same positive/negative selection-based strategy as previous studies to achieve knock-out of the OsNRPD1 gene. The binary vector will carry DsRed and 35s-hph genes as pollen screening and positive selection markers, while T-DNA will carry OsNRPD1 flanking homologous sequences and diphtheria toxin A subunit gene (DT-A) as negative selectable markers. The pollen will be sorted out by FACs and subjected to small RNA deep sequencing and global methylation analysis to identify the small RNAs specifically produced during pollen development and their activity on TE regulation. This study will provide a platform to expand our understanding of small RNAs and DNA methylation-mediated TE regulation and its importance in reproduction and seed development.