Trihelix (TH) proteins are a family of plant-specific transcription factors that play a role in light response and are extensively involved in plant growth and development, as well as in various stress responses. However, the function of TH genes in Brassica napus (B. napus) remains unclear, as does the evolution and differentiation pattern of TH genes in Brassica plants. Here, we identified a total of 455 TH genes in seven species, including six Brassica species and Arabidopsis, which were grouped into five clades, GT-1, GT-2, GTγ, SH4, and SIP1, each with 69, 142, 44, 55, and 145 members, respectively. The types and distributions of motifs of the TH proteins and the structures of the TH genes are conserved in the same subgroup, and some variations in certain amino acid residues occur in B. napus when inheriting motifs from Brassica rapa (B. rapa) and Brassica oleracea (B. oleracea). Collinearity analysis revealed that the massive expansion of TH genes in tetraploid species was attributed to the hetero-tetraploidization of diploid ancestors and gene duplication events within the tetraploid species. Comparative analysis of the membership numbers of five subgroups in different species revealed that the GT-2 and SIP1 genes underwent significant expansion during evolution, possibly to support the better adaptation of plants to their environments. The differential expression of the BnaTH genes under five stresses indicates that the BnaTH genes are involved in plant responses to stresses such as drought, cold, and heat. The presence of different stress-responsive cis-elements in the upstream promoter region of the genes indicated that BnaTH genes have the potential to cope with variable environments. Meanwhile, qRT-PCR analyses also confirmed that five TH genes respond to different abiotic stresses. Our results provide information and candidates for further studies on the role of TH genes in stress resistance of B. napus.

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