Expression profiling of micro RNAs associated with water stress in banana (Musa spp.)

Abstract

The study entitled “Expression profiling of microRNAs associated with water stress in banana (Musa spp.)” was carried out during 2018-2020, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective was to study the expression of computationally predicted miRNAs in banana cultivar Nendran (Musa AAB) under water stress conditions. Five computationally predicted miRNAs were selected from a study previously conducted in the Department of Plant Biotechnology. The miRNAs were selected based on the function of their target genes that have a possible role in stress responses. The miRNAs selected were miR-3900-5p (target: Nitrate transporter 1.1), miR-67 (target: Probable xyloglucan endotransglucosylase/hydrolase protein 33), miR-971-5p (target: 20 kDa chaperonin protein), miR-2293 (target: Putative Zinc finger protein VAR3 chloroplastic), miR-449c-3p (target: ABC transporter C family member 13). Three-months-old in vitro raised banana cv. Nendran plants were subjected to water stress by withdrawing irrigation for one week. Water stress was confirmed by measuring the relative water content in the leaf samples after 7 days. For expression analysis of miRNAs, total RNA was isolated from the leaf samples using the modified Rodrigues-Garcia method and reverse transcribed to cDNA. Stem loop primers were used for miRNAs and PCR was carried out using a specific forward and a universal reverse primer. The expression profile of the miRNAs and their target genes were studied by RT-qPCR. A melt curve analysis was performed to determine the specificity of the products. Reactions were done with three biological replicates and two technical replicates. The results showed an upregulation of miR-2293 (6 to 10-fold) in all the plants. But the expression pattern of its target gene (Putative Zinc finger protein VAR3 chloroplastic gene) showed a reduction in its expression by half, indicating an inverse correlation between the miR-2293 and its predicted target, which in turn indicate a miRNA-target interaction in response to water stress. The miR-67 expression showed a 2 to 4 fold increase in the water stress-imposed plant samples compared to control. Its target, Probable xyloglucan endotransglucosylase/hydrolase protein 33, also showed an increase in its expression (2.5 and 4.5 fold). However, there was no inverse correlation between the miRNA and its target. In RT-qPCR, miR-3900-5p and miR-971-5p did not produce expected amplicons and their target genes did not show any specific pattern of expression in water stress-imposed plants. miR-449c-3p and its target ABC transporter C family member 13 gene did not show expression in any of the samples. The study showed that miR-2293 and miR-67 are water stress responsive in banana cv. Nendran. Further studies on other identified targets may help in understanding the role of these miRNA in water deficient stress conditions.