Sequence And Functional Evolution Of CBF-like Genes In Cruciferous Plants

The generation of new genes or new gene functions is the basises of biological phenotypic variation and plays a significant role in the course of biological evolution.Single gene replication,chromosome fragment replication and whole genome replication can all contribute to the generation of new genes,which are the fundamental cause of gene family formation and expansion.CBF-like genes pertain to DREB subfamily of AP2/ERF transcription factor family which is peculiar in plants,and its encoding transcription factors can characteristically recognize and bond with the CRT/DRE elements on the downstream cold response gene promoter region,and activate gene expression,thereby heightening plant's resistibility to cold(CBF1/2/3)or drought(CBF4)stress or affecting plant growth and development process(DDF1 and DDF2).The team early researched the origin and evolution of CBF-like genes in terrestrial plants systematacially,and found that they has produced differentiation on the number of copies in diverse species.In this study,we selected Carica papaya,Gossypium raimondii and Theobroma cacao as outgroups and representative 9 species of Cruciferae that have obtained full genome sequences,we studied evolution of CBF-like gene sequence and function for further in-depth discussion.The results are summarized as follows:1.Evolution history of CBF-like genes in Cruciferae:CBFs and DDFs have been differentiated in the three outgroups,but only in the Cruciferae,CBFs further differentiated into CBF1/2/3 and CBF4,and DDFs further differentiated into DDF1 and DDF2;In terms of gene copy number,there were both expansion and contraction in the species of Cruciferae.There were 6 copies(CBF1-3+CBF4+DDF12)in 5 species(Arabidopsis thaliana,Arabidopsis lyrata,Capsella grandiflora,Cap.Rubella and Boechera stricta),and there were 10 copies in Brassica rapa.DDF2s in Brassica oleracea capitata were lost,there was only one CBF1?3 in Eutrema salsugineum.In terms of the evolution model after gene duplication,the concerted evolution of CBF1-3 in response to environmental cold signal may have occurred in multiple species.The differentiation of single copy CBF4 and DDF1-2 in each species was consistent with the history of species differentiation,which was a typical divergent evolution.Brassica experienced copy number expansion(Br.rapa)accompanied by genome triploid events,followed by partial copy loss.In addition,there was also gene loss CBFs in E.salsugineum,which belonged to the birth-death evolution pattern after gene duplication.2.Differentiation of CBF protein sequences and transcriptional activation activity:In terms of protein structure,proteins of four branches of Cruciferae plants evolved more abundant motifs,and each branch also evolved its own unique motifs,compared with the three plants of the outgroup.The transcriptional activation activities of all proteins were also differentiated to a certain extent,and the activity loss occurred randomly in the phylogenetic tree,but did not aggregate into a monophyletic line,and there was no genealogical specificity of species or genus.On the phylogenetic branches under positive selection were identified by bioinformatics analysis,the proteins encoded by some genes were also functionally differentiated,indicating that the differentiation of transcriptional activation activities of these proteins was probably driven by natural selection.3.Mutation of a single amino acid in cruciferae plants can result in loss or reduction of transcriptional activation activity of proteins:single-point mutation of amino acids in CBF4 and DDF 1 branches with distinct differentiation of transcriptional activation activity was found to significantly reduce transcriptional activation activity of CBF4 at 6 amino acid sites in AthCBF4 of A.thaliana and 9 amino acid sites in BstCBF4 of Bo.Stricta.The F/I/L mutation at locus 120 in DDF1 is probably responsible for the reduced activity of CruDDF1 in Cap.rubella,BraDDF1L2 in Br.rapa,and EsaDDF1 in E.salsugineum and the cause of the loss of activity AthDDF1 of A.thaliana,AlyDDF1 of A.lyrata and BstDDF1 of Bo.Stricta.