Highly Efficient Method for Construction of Rice Artificial MicroRNA Vectors

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'. Thi...

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Bibliographic Details
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
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Summary: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.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-010-9291-4