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


Ontology type: schema:Chapter     


Chapter Info

DATE

2016

AUTHORS

Basdeo Bhagwat , Ming Chi , Dianwei Han , Haifeng Tang , Guiliang Tang , Yu Xiang

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. More... »

PAGES

149-62

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-1-4939-3393-8_14

DOI

http://dx.doi.org/10.1007/978-1-4939-3393-8_14

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1039774719

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/26843173


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