Browsing by Author "Shruthy, N S"

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    Elucidation of role of small RNA mediated gene regulation in secondary metabolite pathway of black pepper (Piper nigrum L.)
    (Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2024-07-19) Shruthy, N S; Asha, S
    The study entitled “Elucidation of role of small RNA mediated gene regulation in the secondary metabolite pathway of black pepper (Piper nigrum L.)” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2023-2024. Piperine, an amide alkaloid that contributes the unique spicy flavor is the major economic secondary metabolite of black pepper which is renowned for its pharmacological activities and therapeutic efficacies. The biosynthetic pathway of various secondary metabolites was highly influenced by a regulatory factor, microRNA- ‘the master regulators of gene expression’. They are small, non-coding RNAs of typically 18-24 nucleotides and can be a useful tool in metabolic engineering. Therefore, a better understanding of the mode of action of miRNA is very essential to exploit them in improving the plant for enhanced secondary metabolite production. Hence, the present study focuses on ‘identification and characterization of small RNAs involved in piperine biosynthesis from black pepper (Piper nigrum L.) In this study, the microRNAs targeting the key genes involved in the piperine biosynthesis pathway were analyzed by combined in silico-experimental method. From the 19 million reads of small RNA transcriptome, 303 conserved families of MiRNA were reported in black pepper. Among these, potential candidates of small RNAs were predicted to target the key genes involved in piperine biosynthesis such as Piperine synthase, piperamide synthase, and Piperoyl CoA Ligase genes. From these candidates, we characterized conserved and novel microRNAs. Precursor miRNAs for each candidate were also predicted based on their distinctive features such as length, ability to form hairpin stem-loop structure, and minimal folding energy. Among the potential candidate miRNAs, ‘Pni_miR5654’ and novel miRNAs such as ‘Pni_miR19’ and Pni_miR33’ were predicted for experimental validation. The tissue-specific expression of these miRNAs and their cognate targets in different parts of the black pepper variety Panniyur-1 were evaluated. The result indicates that the immature spike has the highest expression of microRNAs compared to leaf and mature berries whereas the highest expression of cognate target genes was observed in mature berries compared to leaf and immature spikes, that is, the miRNA: target pairs were inversely correlated. Further to analyse the expression of miRNA: target pairs in bio-elicitated callus cultures, initially the callus initiated from berries were treated with the culture filtrate of Pirimorsphora indica and R/NA was isolated at 0, 24, and 48 hrs after treatment. The study sowed and upregulation in the expression of both the miRNAs and their cognate pairs in 24 hour treatment. In this present study, the microRNA candidates Pni_miR5654, Pni_miR19’ and ‘Pni_miR33’ were identifies to have gene regulatory role in piperine biosynthesis by targeting the key genes in the pathway. Although miRNA medicated regulation was detected in the spike and berry stages, the inverse correlation of miRNA: target pairs was not found in in vitro cultures of black pepper, indicating the cellular reprogramming of miRNAs during callogenesis. The data generated from this study could be helpful in modifying the target genes, constructing artificial miRNAs, and performing inhibition of miRNA in order to enhance the production of piperine in black pepper.