Barley Transcriptome Analysis and amiRNA Design

Genomic sequences for HvMADS27 and HvMIR444c were obtained from the Ensembl Plants database³ and aligned with the cDNA sequence using MAFFT version 7.⁴ Results from transcriptome sequencing were quality checked using the FASTQC program, and adaptor trimming was performed using Trimmomatic software. All obtained reads were mapped to the barley genome and transcriptome using HISAT2 and SALMON programs, respectively, and statistical analysis was performed in R studio environment using DESeq2 software (Love et al., 2014 (link)). Thereafter, the online tool WMD3-Web MicroRNA Designer⁵ was used to prepare construct overexpressing artificial miRNA (amiRMADS27) that targets HvMADS27 mRNA. Root systems were analyzed using an Epson scanner and WinRHIZO software (Regent Instruments, Inc., Quebec, Canada). Total root length was further analyzed statistically using Statistica software 13.1 (Kraków, Poland). Densitometric analysis of western blots was performed using Image Studio Lite program version 5.2.5, and the amount of BG1 protein was normalized using WT control to normalize all bands (treated as 1.0, LI-COR, United States).

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Smoczynska A., Pacak A., Grabowska A., Bielewicz D., Zadworny M., Singh K., Dolata J., Bajczyk M., Nuc P., Kesy J., Wozniak M., Ratajczak I., Harwood W., Karlowski W.M., Jarmolowski A, & Szweykowska-Kulinska Z. (2022). Excess nitrogen responsive HvMADS27 transcription factor controls barley root architecture by regulating abscisic acid level. Frontiers in Plant Science, 13, 950796.

Publication 2022

WinRHIZO software Image Studio Lite

Barley Cdna Densitometric Genomic Love Mirna Mrna Plants Protein Root Salmon Transcriptome Western blots analysis

Corresponding Organization : Adam Mickiewicz University in Poznań

Other organizations : Institute of Dendrology, Polish Academy of Sciences, Panjab University, Nicolaus Copernicus University, University of Life Sciences in Poznań, John Innes Centre, Norwich Research Park

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Variable analysis

independent variables

Overexpression of artificial miRNA (amiRMADS27) that targets HvMADS27 mRNA

dependent variables

Root system analysis using Epson scanner and WinRHIZO software (total root length)
BG1 protein amount determined by densitometric analysis of western blots

control variables

WT (wild-type) control for BG1 protein normalization

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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