Local gene silencing in plants via synthetic dsRNA and carrier peptide

Quick and facile transient RNA interference (RNAi) is one of the most valuable plant biotechnologies for analysing plant gene functions. To establish a novel double‐strand RNA (dsRNA) delivery system for plants, we developed an ionic complex of synthetic dsRNA with a carrier peptide in which a cell‐penetrating peptide is fused with a polycation sequence as a gene carrier. The dsRNA–peptide complex is 100–300 nm in diameter and positively charged. Infiltration of the complex into intact leaf cells of Arabidopsis thaliana successfully induced rapid and efficient down‐regulation of exogenous and endogenous genes such as yellow fluorescent protein and chalcone synthase. The present method realizes quick and local gene silencing in specific tissues and/or organs in plants.     

dsRNA介导植物基因沉默及其应用

植物双链RNA(double stranded RNA,dsRNA)能有效干扰同源基因的表达,近年来已成为功能基因组学研究上的新方法。本文综述了植物dsRNA介导的转基因沉默现象及其特点、分子作用机制、主要介导方法,以及近年来在植物功能基因组学研究上的应用情况。     

MicroRNA: Biogenesis, Function and Role in Cancer

MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. The conventional biogenesis pathway consists of two cleavage events, one nuclear and one cytoplasmic. However, alternative biogenesis pathways exist that differ in the number of cleavage events and enzymes responsible. How microRNA precursors are sorted to the different pathways is unclear but appears to be determined by the site of origin of the microRNA, its sequence and thermodynamic stability. The regulatory functions of microRNAs are accomplished through the RNA-induced silencing complex (RISC). MicroRNA assembles into RISC, activating the complex to target messenger RNA (mRNA) specified by the microRNA. Various RISC assembly models have been proposed and research continues to explore the mechanism(s) of RISC loading and activation. The degree and nature of the complementarity between the microRNA and target determine the gene silencing mechanism, slicer-dependent mRNA degradation or slicer-independent translation inhibition. Recent evidence indicates that P-bodies are essential for microRNA-mediated gene silencing and that RISC assembly and silencing occurs primarily within P-bodies. The P-body model outlines microRNA sorting and shuttling between specialized P-body compartments that house enzymes required for slicer -dependent and -independent silencing, addressing the reversibility of these silencing mechanisms. Detailed knowledge of the microRNA pathways is essential for understanding their physiological role and the implications associated with dysfunction and dysregulation.     

Geminivirus-based vectors for gene silencing in Arabidopsis

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.