Genome-Wide Identification, Molecular Evolution, and Expression Profiling Analysis of Pectin Methylesterase Inhibitor Genes in Brassica campestris ssp. chinensis

Abstract

Pectin methylesterase inhibitor genes (PMEIs) are a large multigene family and play crucial roles in cell wall modifications in plant growth and development. Here, a comprehensive analysis of the PMEI gene family in Brassicacampestris, an important leaf vegetable, was performed. We identified 100 BrassicacampestrisPMEI genes (BcPMEIs), among which 96 BcPMEIs were unevenly distributed on 10 chromosomes and nine tandem arrays containing 20 BcPMEIs were found. We also detected 80 pairs of syntenic PMEI orthologs. These findings indicated that whole-genome triplication (WGT) and tandem duplication (TD) were the main mechanisms accounting for the current number of BcPMEIs. In evolution, BcPMEIs were retained preferentially and biasedly, consistent with the gene balance hypothesis and two-step theory, respectively. The molecular evolution analysis of BcPMEIs manifested that they evolved through purifying selection and the divergence time is in accordance with the WGT data of B. campestris. To obtain the functional information of BcPMEIs, the expression patterns in five tissues and the cis-elements distributed in promoter regions were investigated. This work can provide a better understanding of the molecular evolution and biological function of PMEIs in B. campestris.

Keywords: Brassica campestris; PMEIs; evolution; expression patterns.

Figure 1

Gene structures and conserved motifs of PMEIs in Brassica campestris. (a) Exon–intron organization of BcPMEIs. The black lines and green boxes stand for introns and exons, respectively; (b) Distributions of conserved motifs in BcPMEIs. The differently colored boxes, numbered 1–10 at the bottom, denote differently conserved motifs.

Figure 2

Chromosomal locations of PMEIs in Brassica campestris. The chromosome numbers are represented on the top of each chromosome. The positive (+) and negative (−) signs indicate forward and reverse orientation, respectively. The names of tandem genes lie next to pound signs (#). Four BcPMEIs could not be mapped onto a specific chromosome.

Figure 3

Syntenic analysis of PMEIs in Brassica campestris and Arabidopsis thaliana. The 24 genomic blocks are colored on the basis of the inferred ancestral chromosomes following an established convention.

Figure 4

The retained rate of PMEIs in Brassica campestris. (a) Retained rates of BcPMEIs, randomly selected genes, and core eukaryotic genes; (b) retention rates by the number of homologous copies in the syntenic region; (c) retained rates of BcPMEIs, randomly selected genes, and core eukaryotic genes among the three subgenomes of B. campestris.

Figure 5

Frequency distributions of the Ks and Ka/Ks values of PMEI homolog pairs. (a,b) Ks and Ka/Ks value distributions of the PMEI ortholog pairs between the Brassica campestris and Arabidopsis genomes; (c,d) Ks and Ka/Ks value distributions of the PMEI paralog pairs in the B. campestris genome. The vertical axes represent the frequency of paired sequences and the horizontal axes represent the Ks and Ka/Ks values at a 0.1 interval.

Figure 6

Hierarchical clustering and heat map representation displaying the expression profiles of BcPMEIs in root (R), stem (St), leaf (L), inflorescence (Inf), and silique (Si). The gene expression levels in the five tissues were explored by qRT-PCR. The scale bars on the left indicate relative expression level. The vertical dark bars on the right depict the seven groups of BcPMEIs. The grey boxes represent the undetectable expression.

Figure 7

GUS staining of the P_PMEI22::GUS inflorescence (a) and P_PME44::GUS inflorescence (b).

Figure 8

Expression profile analysis of representative BcPMEIs by the Illumina RNA-Seq data in A1–A5 and B1–B5. A1–A5 and B1–B5 present the five developmental stages of pollen in “Bcajh97-01A” and “Bcajh97-01B”, respectively.

Figure 9

Expression profile analysis of representative BcPMEIs by the Illumina RNA-Seq data in unpollinated pistils (CK) and pollinated pistils at 1, 3, and 10 h after pollination (HAP).