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Expression Pattern of Plant miRNAs by Classical Transcriptional Fusion Constructs

  • Andrea Tovar-Aguilar
  • Karla A. Sánchez-Elizondo
  • Alejandra Rodríguez-Rodríguez
  • Marcos I. González-Jaime
  • Genaro Patiño-López
  • Vadim Perez-Koldenkova
  • Jesús A. Badillo-Corona
  • Noé V. Durán-FigueroaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

microRNAs are noncoding RNAs of 20–24 nucleotides (nt) in length that act as repressors of genes and are important in key developmental processes in the entire life cycle of plants. To determine the function of a microRNA, the first step is to resolve its expression pattern; this can be achieved by in situ hybridization, RNA blot assays, or quantitative PCR. However, the study of the expression of a MIR gene is straightforward with the use of reporter proteins such as β-D-glucuronidase (GUS), GFP, or mCherry. To do this, it is necessary to clone the promoter region of the MIR gene and place it upstream of the reporter gene; in this way the activity of the promoter will be a direct reflection of the expression of the MIR gene. Here, we indicate step by step how to make transcriptional fusion constructs from the cloning of a promoter region of a MIR gene fused to the classical reporter proteins GUS and mCherry, the latter with codon optimization for better expression in Arabidopsis thaliana. This method is particularly useful to dissect the promoter region of a MIR gene and to find its expression pattern in a tissue and developmental specific manner.

Key words

miRNAs Transcriptional fusion MIR gene promoter Root Arabidopsis 

Notes

Acknowledgments

SIP-IPN 20182227 support research in the laboratory of NVDF. ATA is a PhD student, and KASE, ARR, and MIGJ are Master’s student; all have a scholarship by CONACyT-Mexico.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Andrea Tovar-Aguilar
    • 1
  • Karla A. Sánchez-Elizondo
    • 1
  • Alejandra Rodríguez-Rodríguez
    • 1
  • Marcos I. González-Jaime
    • 1
  • Genaro Patiño-López
    • 2
  • Vadim Perez-Koldenkova
    • 3
  • Jesús A. Badillo-Corona
    • 1
  • Noé V. Durán-Figueroa
    • 1
    Email author
  1. 1.Instituto Politécnico NacionalUnidad Profesional Interdisciplinaria de BiotecnologíaMexico CityMexico
  2. 2.Laboratorio de Investigación en Inmunología y ProteómicaHospital Infantil de México Federico GómezMexico CityMexico
  3. 3.Laboratorio de Microscopía, Centro de Instrumentos, Coordinación de Investigación en Salud, Centro Médico Nacional Siglo XXIInstituto Mexicano del Seguro SocialMexico CityMexico

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