A 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'. 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.
Wang
Xuming
X
Institute of Virology and Biotechnology (MOA and Zhejiang Provincial Key Laboratory for Plant Virology), Zhejiang Academy of Agricultural Sciences, Shiqiao Road 198#, Hangzhou, 310021, Zhejiang, People's Republic of China. wangxuming@gmail.com
Yang
Yong
Y
Yu
Chulang
C
Zhou
Jie
J
Cheng
Ye
Y
Yan
Chengqi
C
Chen
Jianping
J
D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D020868:Gene Silencing; D017343:Genes, Plant; D005822:Genetic Vectors; D035683:MicroRNAs / Q000235:genetics*; D012275:Oryza / Q000235:genetics*; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018744:DNA, Plant; D035683:MicroRNAs