title: Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.
abstract: Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.
authors: Pacific Agri-Food Research Center, Agriculture and Agri-Food Canada, 4200 Highway 97, Box 5000, Summerland, BC, Canada, V0H 1Z0.
authors: Basdeo
authors: B
authors: Bhagwat
authors: Pacific Agri-Food Research Center, Agriculture and Agri-Food Canada, 4200 Highway 97, Box 5000, Summerland, BC, Canada, V0H 1Z0.|College of Forestry, Northwest A & F University, Yangling, Shaanxi, China.
authors: Ming
authors: M
authors: Chi
authors: Department of Computer Science, University of Kentucky, Lexington, KY, 40506, USA.
authors: Dianwei
authors: D
authors: Han
authors: Provincial State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou, 450002, China.
authors: Haifeng
authors: H
authors: Tang
authors: Provincial State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou, 450002, China. gtang1@mtu.edu.|Department of Biological Sciences, Michigan Technological University, 740 Dow Building, Houghton, MI, 49931, USA. gtang1@mtu.edu.
authors: Guiliang
authors: G
authors: Tang
authors: Pacific Agri-Food Research Center, Agriculture and Agri-Food Canada, 4200 Highway 97, Box 5000, Summerland, BC, Canada, V0H 1Z0. yu.xiang@agr.gc.ca.
authors: Yu
authors: Y
authors: Xiang
mesh_terms: D060054:Agrobacterium / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D018749:RNA, Plant
keywords: Agrobacterium-mediated transient expression system; artificial microRNA; Plant amiRNA expression vector; amiRNA design and construction; miR168a