Genome-wide identification and expression analysis of the R2R3-MYB gene family in tobacco (Nicotiana tabacum L.)

Abstract

Background: The R2R3-MYB transcription factor is one of the largest gene families in plants and involved in the regulation of plant development, hormone signal transduction, biotic and abiotic stresses. Tobacco is one of the most important model plants. Therefore, it will be of great significance to investigate the R2R3-MYB gene family and their expression patterns under abiotic stress and senescence in tobacco.

Results: A total of 174 R2R3-MYB genes were identified from tobacco (Nicotiana tabacum L.) genome and were divided into 24 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were especially conserved among the NtR2R3-MYB genes, especially members within the same subgroup. The NtR2R3-MYB genes were distributed on 24 tobacco chromosomes. Analysis of gene duplication events obtained 3 pairs of tandem duplication genes and 62 pairs of segmental duplication genes, suggesting that segmental duplications is the major pattern for R2R3-MYB gene family expansion in tobacco. Cis-regulatory elements of the NtR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponsive. Expression profile analysis showed that NtR2R3-MYB genes were widely expressed in different maturity tobacco leaves, and however, the expression patterns of different members appeared to be diverse. The qRT-PCR analysis of 15 NtR2R3-MYBs confirmed their differential expression under different abiotic stresses (cold, salt and drought), and notably, NtMYB46 was significantly up-regulated under three treatments.

Conclusions: In summary, a genome-wide identification, evolutionary and expression analysis of R2R3-MYB gene family in tobacco were conducted. Our results provided a solid foundation for further biological functional study of NtR2R3-MYB genes in tobacco.

Keywords: Gene expression; Nicotiana tobacum L.; Phylogenetic analysis; R2R3-MYB transcription factors; Stress response.

Fig. 1

The distribution of NtR2R3-MYB genes on 24 chromosomes in tobacco. The scale bar of the left displays the length of tobacco chromosomes

Fig. 2

Gene duplication of NtR2R3-MYB genes on 24 chromosomes of tobacco genome. The segmental duplicated gene pairs are linked by different colored lines, and the tandem duplicated gene pairs of Nt04 are marked with red dashed lines. The corresponding relationships of duplicated gene are listed in Additional file 2: Table S2

Fig. 3

Gene structure and evolution of R2R3-MYB family in Nicotiana tabacum. A Phylogenetic relationships of NtR2R3-MYBs. Different subgroups were marked with different colors. B Intron-exon structure of NtR2R3-MYBs. Green boxes: exons; Yellow boxes: UTR; spaces between the boxes: introns. The scale bar of bottom demonstrates the length of exons and introns

Fig. 4

Conserved motifs for NtR2R3-MYB proteins in Nicotiana tabacum. Different motifs are showed with different colored boxes and numbers (1–20). The gray lines represent the non-conserved sequences. The lengths of motifs can be estimated using the scale at the bottom

Fig. 5

Sequence logos of the conserved R2 and R3 repeats of the NtR2R3-MYB domain. A Sequence logo of R2 in NtR2R3-MYBs. B Sequence logo of R3 in NtR2R3-MYBs. Asterisks indicate the conserved tryptophan residues (Trp) in the NtR2R3-MYB domain, and helix denote the helical structure among NtR2R3-MYBs

Fig. 6

Predicted cis-elements in NtR2R3-MYBs promoters. Different shapes and colors represent the different types of cis-elements. Annotations of cis-elements were listed in Additional file 4: Table S4

Fig. 7

Phylogenetic tree of Nicotiana tabacum and Arabidopsis thaliana R2R3-MYB genes. The phylogenetic relationships were constructed using MEGA-X by the maximum likelihood (ML) method (1000 bootstrap replicates). The amino acid sequences of 174 NtR2R3-MYB and 126 AtR2R3-MYB proteins were used. Arabidopsis and tobacco are marked with yellow circles and green circles respectively

Fig. 8

The expression of 78 NtR2R3-MYBs in tobacco leaves at five senescence stages. FPKM values for NtR2R3-MYB genes were transformed by log10. Red or blue colors represent the difference in expression levels in each sample (M1-M5), respectively

Fig. 9

Expression of 69 NtR2R3-MYB genes in response to cold and slat treatments. RNA-seq data was obtained by using Genevestigator software. Expression values from RNA-seq data were log10-transformed

Fig. 10

Relative expression level of 15 NtR2R3-MYBs in response to cold, salt, and drought treatments. Error bars are standard deviations of three biological replicates. Asterisks were used to indicate the significant degree of the expression level compared to the value of the control (*P < 0.05, **P < 0.01)