Genome-Wide Identification and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cymbidium ensifolium

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the <i>Cymbidium ensifolium</i> genome and divided into 18 subfamilies. Most <i>CebHLHs</i> contain numerous <i>cis-acting</i> elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the <i>CebHLHs</i>, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 <i>CebHLHs</i> were differentially expressed in four different color sepals, especially <i>CebHLH13</i> and <i>CebHLH75</i> of the S7 subfamily. The expression profiles of <i>CebHLH13</i> and <i>CebHLH75</i> in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of <i>CebHLHs</i> in flower color formation.