Genome-Wide Analysis of Myeloblastosis-Related Genes in Brassica napus L. and Positive Modulation of Osmotic Tolerance by BnMRD107

Abstract

Myeloblastosis (MYB)-related transcription factors comprise a large subfamily of the MYB family. They play significant roles in plant development and in stress responses. However, MYB-related proteins have not been comprehensively investigated in rapeseed (Brassica napus L.). In the present study, a genome-wide analysis of MYB-related transcription factors was performed in rapeseed. We identified 251 Brassica napus MYB (BnMYB)-related members, which were divided phylogenetically into five clades. Evolutionary analysis suggested that whole genome duplication and segmental duplication events have played a significant role in the expansion of BnMYB-related gene family. Selective pressure of BnMYB-related genes was estimated using the Ka/Ks ratio, which indicated that BnMYB-related genes underwent strong purifying selection during evolution. In silico analysis showed that various development-associated, phytohormone-responsive, and stress-related cis-acting regulatory elements were enriched in the promoter regions of BnMYB-related genes. Furthermore, MYB-related genes with tissue or organ-specific, stress-responsive expression patterns were identified in B. napus based on temporospatial and abiotic stress expression profiles. Among the stress-responsive MYB-related genes, BnMRD107 was strongly induced by drought stress, and was therefore selected for functional study. Rapeseed seedlings overexpressing BnMRD107 showed improved resistance to osmotic stress. Our findings not only lay a foundation for further functional characterization of BnMYB-related genes, but also provide valuable clues to determine candidate genes for future genetic improvement of B. napus.

Keywords: Brassica napus; MYB-related transcription factors; abiotic stress; expression profiles; phylogenetic analysis.

Figure 1

Phylogenetic analysis of MYB-related proteins using MEGA 7.0 with the neighbor-joining (NJ) method. Numbers at the nodes represent the reliability percentage of the bootstrap values based on 1,000 replications. Red triangles and green circles represent the MYB-related proteins in Arabidopsis thaliana (A. thaliana) and Brassica napus (B. napus), respectively.

Figure 2

Gene structures and motif compositions of MYB-related proteins in B. napus. (A) Exon-intron structures of BnMYB-related proteins. Green boxes indicate 5′- and 3′-untranslated regions (UTR), respectively. Yellow boxes indicate exons, and black lines indicate introns. (B) Distribution of conserved motifs in BnMYB-related proteins. Motifs 1–20 identified by MEME analysis are displayed in different colored boxes.

Figure 3

Amino acid sequence logos of the MYB domains in BnMYB-related proteins. Bit score indicates the information content for each position in the sequence. The conserved Trp (W) residues distributed in the amino acid sequence of MYB domains, and red boxes indicate the conserved motifs. The corresponding clades based on the phylogenetic tree (Figure 2) are indicated on the left for reference.

Figure 4

The Upset plot shows the distributions of homologous MYB-related genes in different rapeseed accessions. The bar chart above represents the number of common homologous genes shared among different rapeseed accessions. The left bar chart at the bottom represents the number of homologous genes in each accession. The right dotted line at the bottom shows the number of accessions contained in the group.

Figure 5

Regulatory cis-elements on the promoter regions of BnMYB-related genes. Locations of different cis-acting elements are arranged on the lines representing the 2 kb upstream region of TSS in the promoters of each BnMYB-related gene. Different colored ovals represent different types of cis-elements.

Figure 6

Temporospatial expression patterns of BnMYB-related genes. Fragments per kilobase of transcript per million mapped reads (FPKM) values of BnMYBR genes are log₂ transformed and are used to represent the gene expression levels. The horizontal axis represents 16 tissues/organs of different developmental stages in rapeseed. The color scale represents gene expression levels with high transcript abundance (red) or low transcript abundance (blue).

Figure 7

BnMYB-related genes in response to four abiotic stress conditions (drought, salt, cold, and heat) and ABA treatment. Ratios of FPKM values under stress conditions to FPKM values under normal conditions are log₂ transformed and used to represent the gene expression levels.

Figure 8

Expression analysis of 13 BnMYB-related genes under (A) drought treatment and (B) cold treatment by qPCR. The expression levels were normalized to BnActin. Data represented the mean ± SE (n =3).

Figure 9

Phenotype of BnMYBR107-OE plants under normal conditions, osmotic stress, and salt treatments. (A) Growth performance of J9712 and BnMYBR107-OE plants under normal conditions, osmotic stress, and salt treatments. (B–D) Hypocotyl length, root length, and fresh weight under normal conditions, osmotic stress, and salt treatments. Data are presented as means ± SD (n = 5). Asterisks (*) indicate significant differences between the transgenic lines and J9712 based on Duncan's multiple range test (*p < 0.05, **p < 0.01).

Figure 10

DAB-staining of cotyledons from J9712 and BnMYBR107-OE plants under normal conditions, osmotic stress, and salt treatments.