| Shoot branching is an important component of plant architecture,which plays important roles in crop canopy structure,cultivation density,and light interception efficiency.Thus,it is closely related to crop production.Brassica napus is one of the most important oil crops in China.However,its multi-branches,large-branch-angle,and altered-branch-height traits result in loose canopy architecture that is not benefit to crop planting density,field management,and harvesting mechanization,thus is not good for B.napus production.Teosinte-branched 1/Cycloidea/Proliferating（TCP）transcription factors consist of a plant-specific transcription factor gene family,which play important roles in the regulation of plant branching development.Therefore,identifying TCP genes in B.napus genome and screening TCP transcription factors that involved in regulating branching development is important for further analyzing the molecular mechanism of candidates in regulating B.napus branching development.The results will promote the B.napus molecular breeding on the basis of optimizing plant architecture.In this study,we performed a gonome-wide identification of the TCP genes in B.napus genome.Then,the phylogenetic and evolutionary mechanism,expression characteristics and protein interaction network of candidate genes were analyzed.We also explored the origin and evolution of TCP gene family in major terrestrial plants,and investigated the origin and evolution mechanism of genes that regulate plant branching development.The candidate genes which involved in regulating B.napus branching were identified,by combined with the above Bioinformatics analyses and the RNA-seq assay.Our study provide key gene resource for further study on the molecular mechanism of candidate TCP genes in regulating B.napus branching development,and provide new strategies for molecular breeding through high-yield ideal plant architecture.The main results of this study are as follows:1.Genome-wide analysis of TCP transcription factor gene family in B.napus.A total of 75 TCP genes were identified in the genome of B.napus,which were divided into two classes（Class I and Class II）with Class II was further divided into CIN and CYC clade according to the phylogenetic analysis.Excepting for eight genes that were located in the unanchored random regions,the remaining 67 TCP genes were distributed unevenly on 17 chromosomes.Gene structure analysis showed that 67%of TCP genes had no intron insertion among the full-length open reading frame（ORF）region.PlantCARE analysis showed that there are many cis-elements involved in plant hormones,light signal response and biotic/abiotic stress response in the promoter region of BnTCPs,suggesting that these genes may respond to these biological processes.Protein sequence analysis showed that excepting for the common conserved TCP domain at the N-terminal of the TCP proteins,four conserved motifs were also found outside the TCP domain.Collinearity analysis showed the whole genome duplication（WGD）event was the main driving force for TCP gene expansion in B.napus,and the TCP genes inherited from Brassica rapa genome tended to be retained in B.napus genome.Protein interaction network analysis showed that TCP members tend to form homo-or hetero-dimer,and their interacting proteins were mainly involved in transcription regulation,RNA metabolism regulation,bud and branch development regulation,and other biological processes.Expression profile analysis showed that the TCP genes in Class I and CIN clade of Class II were widely expressed in different tissues and/or organs at mutiple developmental stages,while the genes in CYC clade only expressed in a few tissues/organs.2.Origin and evolution of TCP genes in land plantsWe identified 535 candidate TCP genes in 49 representative species of aquatic and land plants.We found that the TCP genes only exist in land plants genome,for example,there were two TCP genes in Marchantia polymorpha and five in Physcomitrella patens,indicating that this gene family originated in the early evolution of land plants.And the number of TCP genes in higher plant genome was relatively large,indicating that the TCP genes significantly expanded during plant evolution.Phylogenetic analysis showed that TCP genes in land plants could be divided into two classes（Class I and Class II）.Similarly,Class II could be further divided into CIN and CYC clades.Among them,Class I and CIN clade appeared early in land plant kingdom,while CYC clade was evolved from CIN clade in monocots and eudicots.The evolutionary analysis showed that the WGD event was main force for the expansion of TCP genes in land plants.MiRNA-target analysis showed that miR319 had potential conserved target site in CIN clade of angiosperms,indicating its conservative regulatory role for TCP gene expression.Expression analysis revealed the conserved expression of TCP genes from lower to higher plants.The extensive expression patterns of Class I and CIN clade in different organs or tissues indicate that they were involved in regulating diverse bio-processes.3.The screening of TCP genes involved in regulating B.napus branching developmentBased on the gene structure,expression profile,collinearity,and known functions analyses of TCP genes in 49 plants,we found that the structures and functions of AtBRC1 and its homologous genes are conserved during the evolution.In our phylogenetic tree,there are a total of 15 BnTCPs in the CYC clade.Among which,five genes（BnTCP1,BnTCP12,BnTCP37,BnTCP60 and BnTCP72,BnTCPs）are the homologs of Arabidopsis AtBRC1 gene.Sequence analysis showed that the five BnTCPs proteins and AtBRC1 and its functional characterized homologs in other plants share high sequence homology,with the sequence similarity in their TCP domains was more than 98%.Moreover,the key sites that were involved in DNA binding were highly conserved in these AtBRC1 homologs,indicating that the candidate BnTCPs might bind to similar target genes in B.napus thus having similar regulating role in branching development.Collinearity analysis showed that BnTCP12 was the orthologous gene of AtBRC1,so it was the key candidate gene of BnTCPs,and the next was BnTCP72.Expression analysis showed that the temporal and spatial expression patterns of BnTCP12 and BnTCP72 were similar;BnTCP12 was up-regulated by IAA,6-BA and GA3 treatments while BnTCP72 was obviously up-regulated by 6-BA and GA3treatments.The miRNA analysis showed that BnTCP12 and BnTCP72 may be regulated by mi R5658,thus it may participate in the regulation of branching development in B.napus. |
