Genome-Wide Identification, Classification and Expression Analysis of the MYB Transcription Factor Family in Petunia

Abstract

A lot of researches have been focused on the evolution and function of MYB transcription factors (TFs). For revealing the formation of petunia flower color diversity, MYB gene family in petunia was identified and analyzed. In this study, a total of 155 MYB genes, including 40 1R-MYBs, 106 R2R3-MYBs, 7 R1R2R3-MYBs and 2 4R-MYBs, have been identified in the Petunia axillaris genome. Most R2R3 genes contain three exons and two introns, whereas the number of PaMYB introns varies from 0 to 12. The R2R3-MYB members could be divided into 28 subgroups. Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization, further supporting the results of phylogenetic analysis. Genes in subgroup 10, 11 and 21 were mainly expressed in petal, not in vegetative tissues. Genes in subgroup 9, 19, 25 and 27 expressed in all tissues, but the expression patterns of each gene were different. According to the promoter analysis, five R2R3-MYB and two MYB-related genes contained MBSI cis-element, which was involved in flavonoid biosynthetic regulation. PaMYB100/DPL has been reported to positively regulate to pigmentation. However, although PaMYB82, PaMYB68 and Pa1RMYB36 contained MBSI cis-element, their function in flavonoid biosynthesis has not been revealed. Consistent with existing knowledge, PaMYBs in subgroup 11 had similar function to AtMYBs in subgroup 6, genes in which played an important role in anthocyanin biosynthesis. In addition, PaMYB1 and PaMYB40 belonged to P9 (S7) and were potentially involved in regulation of flavonoid synthesis in petunia vegetative organs. This work provides a comprehensive understanding of the MYB gene family in petunia and lays a significant foundation for future studies on the function and evolution of MYB genes in petunia.

Keywords: MYB family; Petunia hybrida; anthocyanin biosynthesis; gene expression; transcription factor.

Figure 1

Phylogenetic and gene structure analysis of 2R-, 3R- and 4R-MYB genes in Petunia: (A) The phylogenetic tree was constructed with MEGA 10.1.8 using the neighbor-joining (NJ) method with 1000 bootstrap replicates based on a multiple alignment of amino acid sequences of MYB genes. The subgroup was classified and marked by alternated dark and light gray; (B) Protein motif. Schematic diagram of the conserved motifs in the MYB proteins, which were elucidated using MEME. Each motif is represented by a number in the colored box. The black lines represent the non-conserved sequences; (C) Gene structures of PaMYBs. The exons are represented by green box, the black lines connecting two exons represent introns and untranslated regions are indicated by yellow box.

Figure 2

Consensus sequence and the level of conservation of R2R3-MYB domains in Petunia. The sequence logos of the R2 (A) and R3 (B) repeats are based on full-length alignments of petunia R2R3-MYB domains. The bit score indicates the information content for each position in the sequence. Asterisks indicate the conserved residues Trp (W) in the MYB domain.

Figure 3

Phylogenetic tree of R2R3-MYB proteins between Petunia axillaris (P) and Arabidopsis thaliana (S). Neighbor-joining phylogeny was determined by MEGA 10.1.8. The colored shadow marks the subgroups of the R2R3-MYBs.

Figure 4

Regulatory elements in the promoter region of MYB genes in Petunia: (A) Regulatory elements in R2R3-MYB genes promoter. Different colors of box indicate different cis-elements; (B) Regulatory elements in 3R, 4R and 1R-MYB genes promoter.

Figure 5

Tissue-specific gene expression of 19 R2R3-MYB genes of Petunia. The heat map shows the quantitative real-time polymerase chain reaction (qRT-PCR) analysis the expression level of PaMYB genes in in petal, leaf and roots. The gene expression level of tissue with highest expression level was considered as 1. Blocks with colors indicate decreased (blue) or increased (red) transcript accumulation relative to the respective control.

Figure 6

The expression and promoter analysis of 7 PaMYB genes that contained flavonoid biosynthetic regulation elements: (A) Heatmap showed the expression pattern of 5 PaMYBs in different coloration stage of white and purple petunia. S1 indicates budding period, S2 indicates extension period, S3 indicates blooming period; (B) The pictures of white and purple flowers; (C) The distribution of flavonoid biosynthetic regulation elements in 7 MYB genes. Green box indicates the flavonoid biosynthetic regulation elements.

Figure 7

The significantly expressed MYB genes in Petunia in the pigmentation. The color of histogram represents the color of the petal. Each column represents the mean ± SE of three independent experiments each with three replicates. Different capitals indicate significant difference in the same stage. Different lowercases indicate significant difference in different stages.