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Computational Prediction, Target Identification and Experimental Validation of miRNAs from Expressed Sequence Tags in Cannabis sativa. L

Ganesh Selvaraj Duraisamy, Ajay Kumar Mishra, Jernej Jakse and Jaroslav Matousek

MicroRNAs (miRNAs) are approximately 20-22 nucleotide non-coding RNAs, which play an important role in posttranscriptional degradation of target mRNA or inhibition of protein synthesis through binding the specific sites of target mRNA. miRNAs have been extensively studied in various plant species, however, there is no miRNA identified in Cannabis sativa, a crop which has long been used for hemp fibre, for seed and seed oils, for medicinal purposes, and as a recreational drug. In this study, a computational based approach was used to identify and characterize C. sativa miRNAs. A total of 7 miRNAs belonging to 3 miRNA families were identified in cannabis based on homolog search and series of filtering criteria. The identified miRNAs were validated by endpoint PCR and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), confirmed the existence of conserved miRNAs in C. sativa. Based on near-perfect complementarity between cannabis miRNAs and their target mRNA gene sequence, a total of 23 miRNAs targets were identified involved in processes such as plant development, signal transduction, secondary metabolite production, protein degradation, response to environmental stress and pathogen invasion, and ability to regulate their own biogenesis. The cisregulatory elements relevant to biotic and abiotic stress, plant hormone response flavonoid and cannabinoids biosynthesis were identified in the promoter regions of those miRNA genes. Overall, findings from this study will accelerate the way for further researches of miRNAs and their functions in C. sativa, particularly cannabinoid metabolic pathway