TY - CHAP
T1 - Expression pattern of plant miRNAs by classical transcriptional fusion constructs
AU - Tovar-Aguilar, Andrea
AU - Sánchez-Elizondo, Karla A.
AU - Rodríguez-Rodríguez, Alejandra
AU - González-Jaime, Marcos I.
AU - Patiño-López, Genaro
AU - Perez-Koldenkova, Vadim
AU - Badillo-Corona, Jesús A.
AU - Durán-Figueroa, Noé V.
N1 - Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2019.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - Arabidopsis
KW - MIR gene promoter
KW - Root
KW - Transcriptional fusion
KW - miRNAs
UR - http://www.scopus.com/inward/record.url?scp=85060941907&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-9042-9_13
DO - 10.1007/978-1-4939-9042-9_13
M3 - Capítulo
C2 - 30701500
AN - SCOPUS:85060941907
T3 - Methods in Molecular Biology
SP - 175
EP - 185
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -