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Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis)

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Abstract

Basic helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotic organisms and are thought to be one of the largest families of regulatory proteins. This important family of transcriptional regulators plays crucial roles in plant development. However, a systematic analysis of the bHLH transcription factor family has not been reported in Chinese cabbage. In this study, 230 bHLH transcription factors were identified from the whole Chinese cabbage genome and compared with proteins from other representative plants, fungi and metazoans. The Chinese cabbage bHLH (BrabHLH) gene family could be classified into 24 subfamilies. Phylogenetic analysis of BrabHLHs along with bHLHs from Arabidopsis and rice indicated 26 subfamilies. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns and interaction networks of BrabHLHs were analyzed. Distribution mapping showed that BrabHLHs were non-randomly located on the ten Chinese cabbage chromosomes. One hundred and twenty-four orthologous bHLH genes were identified between Chinese cabbage and Arabidopsis, and the interaction networks of the orthologous genes were constructed in Chinese cabbage. Quantitative RT-PCR analysis showed that expressions of BrabHLH genes varied widely under different abiotic stress treatments for different times. Thus, this comprehensive analysis of BrabHLHs represents a rich resource, aiding the elucidation of the roles of bHLH family members in plant growth and development. Furthermore, the comparative genomics analysis deepened our understanding of the evolution of this gene family after a polyploidy event.

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Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program, No. 2012AA100101), China Agriculture Research System CARS-25-A-12, the Fundamental Research Funds for the Central Universities of China (KYZ201111), and a Project Funded by the Priority Academic Program Development of Jiansu Higher Education Institutions.

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Correspondence to Xi-Lin Hou.

Additional information

Communicated by Y. Van de Peer.

Electronic supplementary material

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438_2013_791_MOESM1_ESM.eps

Supplementary Figure 1 Alignment of all the bHLH domain of Chinese cabbage proteins. Shown at the top are the boundaries used in this study to distinguish the DNA-binding basic region, the two a-helixes and the variable loop region. (EPS 7155 kb)

438_2013_791_MOESM2_ESM.eps

Supplementary Figure 2 Alignment of all the bHLH domain of Arabidopsis proteins. Shown at the top are the boundaries used in this study to distinguish the DNA-binding basic region, the two a-helixes and the variable loop region. (EPS 5271 kb)

438_2013_791_MOESM3_ESM.eps

Supplementary Figure 3 Alignment of all the bHLH domain of rice proteins. Shown at the top are the boundaries used in this study to distinguish the DNA-binding basic region, the two a-helixes and the variable loop region. (EPS 5598 kb)

438_2013_791_MOESM4_ESM.eps

Supplementary Figure 4 The predicted DNA-binding characteristics of the bHLH domains of BrabHLH, AthbHLH and OsabHLH proteins. (EPS 5593 kb)

438_2013_791_MOESM5_ESM.eps

Supplementary Figure 5 Alignment of all the bHLH domain of Chinese cabbage, Arabidopsis and rice proteins. Shown at the top are the boundaries used in this study to distinguish the DNA-binding basic region, the two a-helixes and the variable loop region. (EPS 15641 kb)

438_2013_791_MOESM6_ESM.eps

Supplementary Figure 6 Logos of BrabHLH protein motifs. Logos are a visualization tool for motifs. The height of a letter indicates its relative frequency at the given position. (EPS 4580 kb)

438_2013_791_MOESM7_ESM.eps

Supplementary Figure 7 The NJ phylogenetic tree and BrabHLH gene structure of Chinese cabbage. The yellow blocks indicate introns and the green blocks indicate exons. The bHLH domains are shown as red blocks. (EPS 7626 kb)

438_2013_791_MOESM8_ESM.eps

Supplementary Figure 8 The NJ phylogenetic tree of the bHLH domain in plants, metazoans and fungi. (a) The NJ phylogenetic tree of the 3,242 bHLH protein domain in 25 representative plants. The phylogenetic classification was mainly according to Pires and Dolan (2010). (b) The NJ phylogenetic tree of the 698 bHLH protein domain in 12 representative metazoans. The phylogenetic classification was mainly according to a previous report (Ledent et al. 2002). (c) The NJ phylogenetic tree of the 3,948 bHLH protein domain in all the plants, metazoans and fungi used in this study. (d) The NJ phylogenetic tree of the 188 bHLH protein domain in the blended group. This group contained several bHLH proteins of plants, metazoans and fungi. (EPS 13901 kb)

438_2013_791_MOESM9_ESM.eps

Supplementary Figure 9 The relative expression levels of the significantly upregulated bHLH genes during (A) cold, (B) ABA, (c) PEG and (d) GA treatments (relative expression level > 5). The expression value was calculated using the 2−ΔΔCT method. Error bars represent standard errors from three independent replicates. (EPS 6475 kb)

438_2013_791_MOESM10_ESM.eps

Supplementary Figure 10 The NJ phylogenetic tree and the schematic diagram of amino acid motifs of bHLH proteins in Chinese cabbage. (EPS 8263 kb)

Supplementary material 10 (XLS 246 kb)

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Song, XM., Huang, ZN., Duan, WK. et al. Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis). Mol Genet Genomics 289, 77–91 (2014). https://doi.org/10.1007/s00438-013-0791-3

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