关键词: 苦荞, ARF基因家族, 生长素, 株高, 基因表达

Abstract:

【Objective】 The objective of this study is to lay a foundation for the further functional studies and application of ARF genes by identifying the ARF gene family in tartary buckwheat (Fagopyrum tataricum), and analysis of gene structure, conserved domains, phylogeny, tissue expression characteristics, and gene expression levels under exogenous auxin treatment. 【Method】 The data of transcriptome and ARF conservative domains (PF06507) were analyzed to screen for ARF family members in tartary buckwheat. TBtools software was used to analyze the gene structure, NCBI and MEME were used to predict the conserved domain and motif of ARF proteins online, and MEGA X was used to construct ARF protein phylogenetic trees for tartary buckwheat, Arabidopsis thaliana, Oryza sativa, Fagopyrum esculentum, Beta vulgaris, and Glycine max. The FPKM values of six tissues of roots, stems, leaves, flowers, green grains, and black grains were analyzed in the transcriptome data of tartary buckwheat. Heat maps of FtARFs were drawn using TBtools HeatMap to analyze the tissue expression specificity of FtARFs. To predict the cis-acting elements of the FtARFs promoter specifically expressed on the stem, the PlantCARE online website was used. Two parts of tall buckwheat (ZNQ189 and PI673849) and two parts of dwarf buckwheat (PI658429 and PI647612) were treated with 0.5 mg·L ^-1 IAA and the elongation characteristics of the hypocotyls were analyzed. Samples were taken at different time periods (0, 0.5, 1, 6, 12, 24, and 48 h) after auxin treatment, and the expression differences of FtARFs in different hypocotyls were analyzed by qRT-PCR. Moreover, paraffin sections of four materials grown for 7 days were saffron and green stained, and the cell length was measured. 【Result】Total of 26 FtARFs were identified in the buckwheat genome. Chromosome mapping analysis showed that in addition to chromosome 4, FtARFs were distributed in other chromosomes. Analysis of physical and chemical properties showed that amino acid residues ranged from 331 to 1 083 aa and the theoretical isoelectric point ranged from 5.34 to 8.63. Conserved motif analysis identified several differences in motif composition among different groups. Gene structure analysis showed that the number of FtARFs exons ranged from 2 to 15, showing large variation. Phylogenetic analysis showed that 114 ARF proteins were divided into four groups (Group Ⅰ to Group Ⅳ), and FtARFs were distributed throughout all groups. The results of tissue-specific analysis showed that FPKM values of FtARFs differed significantly in different tissues. In roots, stems, and flowers, seven, nine, and four genes showed high expression, respectively, whereas in leaves, green grains, and black grains, the expression values remained low. When exogenous auxin was applied to these four buckwheat materials, the trend of hypocotyl elongation differed, which is consistent with observed changes in cell length. qRT-PCR showed that the expression of FtARFs was higher during the early stage (0.5-1 h) of auxin treatment, and decreased during later stages. When buckwheat seedlings were treated for 0.5 h, the expression of most genes was induced. 【Conclusion】The tartary buckwheat ARF gene structures and protein motifs show diversity among groups and conservation within groups. FtARFs show tissue expression specificity, and nine FtARFs, that are specifically expressed in the stem, respond to IAA induction. These exert a regulatory role in the stem elongation of tartary buckwheat.

Key words: tartary buckwheat, ARF gene family, auxin, plant height, gene expression