A highly efficient method for construction of rice artificial MicroRNA vectors

Mol Biotechnol. 2010 Nov;46(3):211-8. doi: 10.1007/s12033-010-9291-4.

Abstract

Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion of a 'vector modification fragment'. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose 'Highly Efficient gene Silencing Compatible vector' (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient, and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current method, making it particularly suitable for use in a systems biology approach to functional genomic research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers
  • DNA, Plant / genetics
  • Gene Silencing
  • Genes, Plant
  • Genetic Vectors*
  • MicroRNAs / genetics*
  • Oryza / genetics*
  • Phosphorylation
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • DNA Primers
  • DNA, Plant
  • MicroRNAs