Overexpressing drought-responsive miRNA 156 in rice (Oryza sativa L.) enhances drought stress resistance

Abstract

Globally the earth is facing an extreme climate changes including flood and high temperatures, changing the earths surface and weather rapidly. These rapid changes cause a remarkable reduction in plant growth and productivity. The discovery of micro RNA in plant has open a new field of study in plant biotechnology. MicroRNAs play a vital role in abiotic stress and crop improvement by regulating target gene expression. MicroRNA156 (miR156) is a highly conserved and emerging tool for improving various plant traits including crop productivity and drought stress tolerance. The aim of this study was to investigate how miR156 genes interacts with target genes and induce drought stress tolerance. To analyze its role, we constructed overexpressing miR156 lines and knockout lines by using CRISPR/Cas9 system. The sequencing analysis, however, showed no mutation in mature microRNA sequence in both miR156d and miR156g Cas9-mediated mutation lines. As a result, we proceeded research with only overexpressing miR156d and miR156g lines. We observed overexpressing miR156 plants had more tolerant to drought stress than nontransgenic with the higher survival rate and Fv/Fm value. Next, we predicted some targets and screened genes showing antagonic expression pattern to miR156d and miR156g by qRT-PCR. Our highest-ranking candidates include SPL2, SPL17, SPL18, 01t0187200-01 and 09t0513100-01. Based on these results, it can be concluded that overexpressing miRNA156d and miR156g genes improve rice drought tolerance by interacting with its target genes. Finally, it is important to study the interaction between miRNA156 and SPL during flower induction and seed development and needed to address their role in cell number and size during early growth stages. This study provides a strong background for plant biotechnology and crop improvements and offer an important source for further functional analysis and molecular breeding for drought stress tolerance in rice.