| Domains of unknown function(DUFs)are a large sum of protein families whose functions need to be elucidated in eukaryotes.With the development of research,some DUFs have been found to play important roles in growth and development regulation and abiotic stress response of plants.Among them,DUF677 family is an unknown functional protein family specifically in plants.In this study,multiple bioinformatics analysis methods were used to predict the structural characteristics and functions of the OsDUF677 family.The OsDUF677.2(OsU496A)rice lines edited by CRISPR/Cas9 were used as experimental materials to study the function of OsU496A.The main results are as follows:(1)Seven OsDUF677 family members(OsDUF677.1-OsDUF677.7)were divided into three groups according to the phylogenetic tree.OsDUF677.2 and OsDUF677.6 in group I,and OsDUF677.4 and OsD UF677.7 in group Ⅲ were highly expressed in rice grains and roots.Analysis of gene co-expression networks indicated that the OsDUF677 family genes might play important roles in the regulation of grain development and response to abiotic stress.Further protein interaction networks showed that OsDUF677.2,OsDUF677.4,OsDUF677.6 and OsDUF677.7 might be involved in the maintenance of Na+ and K+ homeostasis under salt stress,root architecture,and hormone signaling pathways.Based on the results of bioinformatics analysis,OsDUF677 family proteins may play important roles in root development,hormone regulation and stress response of rice.(2)Compared with wild type(WT),the plant height,flag leaf length and panicle length of OsU496A editing rice lines were significantly reduced.The number of effective tillers and the number of grains per panicle of OsU496A editing rice lines also showed a significant decrease trend.The study on grain traits showed that the enlargement of epidermal cells in the glume of the OSU496A editing rice lines led to the increase of grain length and width,which resulted in a significantly heavier 1000-grain weight than that of the WT,which revealed that OsU496A mainly affected rice yield by regulating the grain development of rice.(3)The total root length and the number of root tip of the Os U496A editing rice seedlings were significantly less than those of the WT.Further analysis of cell morphology in meristem,elongation and mature zones of seedling roots showed that the length and width of meristem cells in OsU496A editing rice lines were significantly reduced compared with WT,and the number of cells in the meristematic zone was also reduced.The width of cells in elongation and mature zones were also significantly reduced.The changes of root phenotype and cell morphology indicated that OsU496A might be involved in the growth of rice roots,and may affect root growth through positively regulation of cell growth.(4)The endogenous IAA levels in the roots of u496a-74 and u496a-79 seedlings were significantly higher than that of the WT.Exogenous NAA treatments showed that the number of roots,total root length,longest root length and other morphological indexes of OsU496A editing rice lines were promoted when treated with lower concentrations of NAA.These results indicated that the optimal concentration of NAA were lower for OsU496A editing rice lines compared with WT,and the OsU496A editing rice lines were more sensitive to NAA treatment Therefore,OsU496A may negative regulate the growth and development of rice roots regulated by auxin.(5)The subcellular localization of OsU496A and E3 ubiquitin ligase OsARI2 showed that OsU496A was located in the cell membrane and nucleus,and OsARI2 was located in the nucleus.These results suggest that OsU496A and OsARI2 may overlapped in spatial localization.In conclusion,as a member of the OsDUF677 family,OsU496A is likely to be involved in the regulation of rice grain development and seedling root growth.These results provide insights into the function of OsU496A,and provide plant materials for exploring the mechanism of grain development and IAA regulating root development in rice. |
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