Highly Efficient Method for Construction of Rice Artificial MicroRNA Vectors

• Published in: Molecular biotechnology 2010-11, Vol.46 (3), p.211-218
• Main Authors: Wang, Xuming, Yang, Yong, Yu, Chulang, Zhou, Jie, Cheng, Ye, Yan, Chengqi, Chen, Jianping
• Format: Article
• Language: English
• Subjects: Base Sequence; Biochemistry; Biological and medical sciences; Biological Techniques; Biotechnology; Cell Biology; Chemistry; Chemistry and Materials Science; DNA Primers; DNA, Plant - genetics; Fundamental and applied biological sciences. Psychology; Gene Silencing; Genes, Plant; Genetic Vectors; Human Genetics; MicroRNAs - genetics; Molecular biology; Oryza - genetics; Oryza sativa; Phosphorylation; Polymerase chain reaction; Protein Science; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; Rice; RNA
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-010-9291-4

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.