Life Sciences & Biotechnology
Title : | A novel miRNA-target module controlling rice grain size: Identification, confirmation and elucidation of its role in grain development and other seed-related traits, targeting crop improvement |
Area of research : | Life Sciences & Biotechnology |
Principal Investigator : | Dr. Pinky Agarwal, National Institute Of Plant Genome Research (NIPGR), New Delhi |
Timeline Start Year : | 2022 |
Timeline End Year : | 2025 |
Contact info : | pinky.agarwal@gmail.com |
Equipments : | Rice grain analyzer
Gel apparatus with accessories and power supply |
Details
Executive Summary : | The project aims to generate transgene free CRISPR/Cas9 edited rice plants which have an increased grain size. This will be done by elucidating a novel miRNA-target module, which has a distinct role in regulation of rice grain size and other seed-related traits, such as starch and protein content. miRNAs are emerging as master regulators controlling plant development and need to be explored in detail. Our previous comparison of transcriptome and miRNome from two rice genotypes with contrasting grain size, resulted in eight miRNA-target modules with contrasting expression between the two genotypes; and ten modules which are differentially expressed throughout grain development in both genotypes. On the basis of these, a hypothesis has been generated that at least one of these modules should have a significantly positive effect on rice grain size. Since grain size and other traits, such as starch and protein content, are related, the module should be able to alter these traits as well. For the elucidation of such a module, foremost is the validation of the expression patterns of miRNAs; and their precursors and targets by qRT-PCR and stem-loop RT-PCR, respectively. This will be followed by establishment of cleavage site in the target by 5′ RLM RACE. Further, the cleavage of target by miRNA will be confirmed in planta to ascertain miRNA activity. Once confirmed, a miRNA-target module will be selected. Various types of rice transgenic plants will be raised to understand the functionality of miRNA and its target. For understanding miRNA function, the miRNA encoding gene will be over expressed; and edited by CRISPR/Cas9 technology. Short tandem target mimic rice plants will also be raised to understand the effect of loss of miRNA function. On the target side, the gene will be over expressed in rice plants. It will also be knocked out/down, as the case may be. Rice plants with a cleavage-resistant target will also be generated by CRISPR/Cas9 technology. All plants generated will be analyzed for their phenotype, especially grain-related traits. This will include grain length, width, weight, starch and protein contents. If the miRNA-target module is effective, plants where the miRNA activity is increased should have a phenotype similar to the ones where target activity is decreased, and vice versa. This will confirm the activity of the miRNA-target module and its role in rice grain development. Any sequence polymorphism at the target site will be subsequently assessed to examine if a natural variation affects the activity of the module. This proposal is a step forward in addressing the global issues of health and food security, aimed at not only in our country but also in the United Nations sustainable development goals. |
Total Budget (INR): | 57,31,203 |
Organizations involved