Strategy to design and functionally analyze an miR targeted reporter gene construct using In planta assay

Plants, upon exposure to environmental stress, express sophisticated and co-ordinated responses to reprogram interconnected defense networks and metabolic pathways. These responses are governed by intricate molecular and biochemical signal transduction processes, which act in harmony to determine tolerance and sensitivity at a holistic level. Modern studies in plant stress biology include identification of key genes that influence stress tolerance and the verification of gene functions using knockout mutants or over-expression lines. Abiotic stresses induce aberrant expression of many miRNAs, suggesting that miRNAs could be a new target for genetically improving plant stress tolerance. MicroRNAs (miRNAs), 21-24 nts in length, are an extensive class of endogenous small RNA molecules that provide a fascinating option for engineering tolerance to environmental stress by serving as non-protein coding switches of both desirable and undesirable gene expressions. During abiotic stress, miRNAs function by regulatingthe stress inducing networks like signalling components and a variety of transcription factors (TFs) that leads to cleavage of the target genes. It is important to identify the expression domains of miRs and their targets to understand how the spatio-temporal regulations are co-ordinated under stress. In this project, we aim to design reporter gene construct containing the target site of selected miR. We have selected pCAMBIA1302 vector with the aim to insert the target site before the ATG of GFP gene such that the coding frame remains intact. Later, the functionality of the designed construct can be checked by expressing the constructs In planta using Agroinfiltration and the technology is expected to greatly favour in molecular pharming.