A highly efficient method for construction of rice artificial MicroRNA vectors

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.