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Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System

  • Basdeo Bhagwat
  • Ming Chi
  • Dianwei Han
  • Haifeng Tang
  • Guiliang TangEmail author
  • Yu XiangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1405)

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.

Key words

Agrobacterium-mediated transient expression system; artificial microRNA amiRNA design and construction miR168a Plant amiRNA expression vector 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Pacific Agri-Food Research CenterAgriculture and Agri-Food CanadaSummerlandCanada
  2. 2.College of ForestryNorthwest A & F UniversityYanglingChina
  3. 3.Department of Computer ScienceUniversity of KentuckyLexingtonUSA
  4. 4.Provincial State Key Laboratory of Wheat and Maize Crop ScienceHenan Agricultural UniversityZhengzhouChina
  5. 5.Department of Biological SciencesMichigan Technological UniversityHoughtonUSA

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