Computational prediction, identification, and expression profiling of microRNAs in banana (Musa spp.) during soil moisture deficit stress

Abstract

Soil moisture deficit stress is one of the most important abiotic stresses limiting the production and productivity of banana (Musa spp.). Plant responses to drought stress are regulated at both the transcriptional and post-transcriptional levels. As post-transcriptional gene regulators, microRNAs (miRNAs) regulate gene expression during drought stress. The present study aimed to identify drought-responsive miRNAs in an expressed sequence tag (EST) library of drought-stressed banana and to study their expression in a drought-tolerant banana cultivar, ‘Saba’, subjected to soil moisture deficit stress. In silico prediction resulted in the identification of three miRNAs (miR156, miR169, and miR2118) whose expression was confirmed using real-time qPCR assays with the respective complementary DNAs (cDNAs) synthesised in multiplex stem-loop-primed reverse-transcription reactions. The selection of a suitable reference miRNA for this study was carried out using six genes, which included the traditional Musa reference gene for 25S rRNA. miR399 was selected as the reference miRNA, based on its constant level of expression in various tissues and sample types. Additional regulatory roles for miR169 on expression of the dehydrin and aquaporin genes in banana were investigated using qPCR assays in drought-stressed leaf samples collected at different times during stress. The results suggested that miR169, miR156, and miR2118 were up-regulated during soil moisture deficit stress, and also that miR169 had an indirect, transcription-level role in regulating dehydrin and aquaporin gene expression. The aquaporin gene exhibited more prominent expression compared to dehydrin in banana during drought stress.