Expression profiling of water stress responsive miroRNAs in banana (Musa spp.)

Abstract

The study entitled “Expression profiling of water stress responsive microRNAs in Banana (Musa spp)” was conducted during 2019-2021, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The study envisaged expression profiling of selected computationally predicted miRNAs in banana cultivar Nendran (Musa AAB) under water stress conditions. Fifty-two novel miRNAs and their targets have been computationally predicted in banana genome A, using NOVOMIR (Mathew, 2018) in a previous study conducted in the Department of Plant Biotechnology. Among five miRNAs were selected in this study for their validation and identifying their association with water stress response. The microRNAs selected are miR-3900-5p (target: Heat shock protein, HSP gene), miR-2172-5p (target: Putative Ethylene Responsive Transcription factor 1 (ERT) gene), miR-971-5p (target: Argonaute, (ARG) gene, miR-6928-5p (target: FAD dependent oxidoreductase gene), and miR-2172-5p (target: Diacylglycerol, DAG gene). Six months old in-vitro raised banana plants of cv. Nendran (Musa AAB) were used in this study. Water stress was induced in the potted plants by withholding irrigation for seven consecutive days. After 7 days, the relative water content in the leaf samples was reduced from 93.84 to 59.32, indicating water stress. The expression profile of the miRNAs and their target genes in banana plants under water stress conditions was analysed by performing Real-time quantitative PCR (RT-qPCR). Total RNA was extracted from leaf samples using the modified Rodrigues Garcia method and reverse transcribed to cDNA using the miRNA specific stem-loop primers designed. Selected miRNAs were amplified using miRNA specific forward and universal reverse primers. The target genes were also amplified by designing primers. The specificity of the primers was determined using a melt curve analysis. All the reactions are conducted with three biological and two technical replicates. Out of the four, three miRNAs (miR-2172-5p, miR-6928-5p and miR-971 5p) and their target genes showed amplification in all the samples. All the water stressed 70 plants showed upregulation of miR-2712-5p (4 to 11fold), and downregulation of miR971-5p (0.1 to 0.4fold) and miR-6928-5p (0.2 to 0.7fold). The two targets of miR2712-5p showed differential expression under the water stressed conditions, DAG gene was downregulated (0.1 to 0.2folds) and ERT gene was upregulated (2 to 3folds). Expression of FAD dependant oxidorductase (The target of miR-6928-5p) and ARG (the target of miR971-5p) was increased up to 3 to 4folds respectively in water stress conditions. Relative gene expression analysis in the water-stressed plants indicated an inverse correlation between all the three miRNAs and their targets, suggesting their strong miRNA: target relation. The study showed that miR-2172-5p, miR-971-5p, miR-6928-5p are water stress-responsive in banana cv. Nendran. Since the products of all the target genes are related to stress responses in plants, an in-depth study of these miRNAs may help develop strategies for water stress management in bananas.