Genome-wide characterization of the DUF966 gene family in rice and functional analysis of OsDSR6 via overexpression

We previously identified seven members of the domain of unknown function 966 (DUF966) family in rice and showed that OsDSR2 and OsDSR3 regulate tolerance to drought, salinity and alkali stresses. However, bioinformatic characterization of this family remains limited, and the biological functions of the remaining members are largely unexplored. In this study, we performed a systematic bioinformatic analysis of the structure and potential function of the rice DUF966 family and focused on the unidentified member OsDSR6 in salinity tolerance. Phylogenetic and collinearity analyses showed that DUF966 genes are more evolutionarily conserved in monocot species. Promoter analysis indicated that family members commonly contain light-responsive elements, abscisic acid-responsive elements (ABREs) and methyl jasmonate (MeJA)-responsive elements. Transcriptional regulatory prediction identified 25 putative transcription factors and 91 microRNA-target interactions associated with this family. Among them, NIGT1 (LOC_Os02g22020) and LOC_Os05g37730 were predicted to bind the promoter regions of OsDSR3 and OsDSR7, respectively, OsDSR6 was predicted to be regulated by osa-miR399a/b/c. OsDSR6 exhibited high expression levels in rice leaves and leaf sheaths. Its expression was rapidly downregulated by salt and drought stress but upregulated by abscisic acid (ABA) treatment. The OsDSR6-encoded protein is localized to the plasma membrane. Functional analysis showed that overexpression of OsDSR6 increases rice sensitivity to salt stress, and the transcript levels of stress-responsive genes, including Lea3, Rab16c, P5CS and OsNCED4, were decreased in OsDSR6-overexpressing plants. Collectively, these findings extend current understanding of the rice DUF966 gene family and identify OsDSR6 as a potential target for molecular breeding of salt-tolerant rice.