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Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco ( Nicotiana Tabacum L.)

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Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco ( Nicotiana Tabacum L.)

Ge Bai et al. Int J Mol Sci.

Abstract

MADS-box genes play a pivotal role in various processes, including floral and seed development, controlling flowering time, regulation of fruits ripening, and respond to abiotic and biotic stressors in planta. Tobacco (Nicotiana tabacum) has been widely used as a model plant for analyzing the gene function, however, there has been less information on the regulation of flowering, and the associated genes. In the present study, a total of 168 NtMADS-box genes were identified from tobacco, and their phylogenetic relationship, chromosome locations, and gene structures were further analyzed. NtMADS-box genes can be clustered into four sub-families of Mα, Mγ, MIKC*, and MIKCC. A total of 111 NtMADS-box genes were distributed on 20 chromosomes, and 57 NtMADS-box genes were located on the unanchored scaffolds due to the complex and incomplete assembly of the tobacco genome. Expression profiles of NtMADS-box genes by microarray from 23 different tissues indicated that members in different NtMADS-box gene subfamilies might play specific roles in the growth and flower development, and the transcript levels of 24 NtMADS-box genes were confirmed by quantitative real-time PCR. Importantly, overexpressed NtSOC1/NtMADS133 could promote early flowering and dwarfism in transgenic tobacco plants. Therefore, our findings provide insights on the characterization of NtMADS-box genes to further study their functions in plant development.

Keywords: MADS-box; Nicotiana tabacum; floral organ; flower development; genome-wide analysis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phylogenetic analysis of NtMADS-box proteins from Arabidopsis and cultivated tobacco. A total of 107 AtMADS-box proteins from Arabidopsis (Arabidopsis thaliana) and 168 NtMADS-box proteins from cultivated tobacco were used to generate the unrooted neighbor-joining (NJ) tree with 1000 bootstrap replicates. The MADS-box proteins are classified into five subfamilies (marked as Mα, Mβ, Mγ, MIKC*, and MIKCC), and distinguished by different colors: NtMADS-box proteins are labeled in a solid circle, and AtMADS-box proteins are labeled in a hollow circle.
Figure 2
Figure 2
Gene structure of the NtMADS-box gene family in tobacco. Exon–intron analyses of identical tobacco NtMADS-box genes were performed with GSDS 2.0. Exons and introns are represented by black rectangle and black lines, respectively. The lengths of exons and introns for each tobacco NtMADS-box gene are shown proportionally.
Figure 3
Figure 3
Conserved motifs of MADS-box genes in tobacco and Arabidopsis were predicted by MEME. Grey lines represent the non-conserved sequences, and ten conserved motifs are indicated by different colors with numbered boxes.
Figure 4
Figure 4
Collinear analysis of the NtMADS-box gene family in tobacco. The annulus represents the tobacco chromosomes, and the scale on the annulus is labeled in megabases (Mb). Homologous genes are linked by the lines. The figure was generated and modified using the Circos program.
Figure 5
Figure 5
Expression profiles of 168 NtMADS-box genes in tissues at different developmental stages. The relative transcript abundances of 168 NtMADS-box were examined via microarray and visualized as a heatmap. The expression profiles of NtMADS-box genes in the 23 different samples, including dry seeds, germination seeds, cotyledons, leaves from two-true leaf stage (labeled as two true leaf_leaf), roots from two-true leaf stage (two true leaf_root), leaves from four-true leaf stage (four true leaf_leaf), roots from four-true leaf stage (four true leaf_root), leaves from six-true leaf stage (six true leaf_leaf), roots from six-true leaf stage (six true leaf_root), leaves from ten-true leaf stage (ten ture leaf_leaf), roots from ten-true leaf stage (ten ture leaf_root), and squaring stage (sepal, fibrous root, and flower), vein, ovary, filament, style, corolla, calyx, stigma, and anther. The X axis is the samples in tissues at different developmental stages. The color scale represents Log2 expression values. The symbol of the star in the MIKCC subfamily represents selected genes for confirming the gene expression by qPCR. Three independent biological experiments with four individual plants were collected for RNA extraction.
Figure 6
Figure 6
Expression profiles of 24 selected NtMADS-box genes in tobacco. The relative transcript abundances of 24 selected NtMADS-box genes were examined via qPCR and visualized as a histogram. The tobacco flowers (120 day old plants) and 6–7 week old seedlings grown in the soil were collected. Three independent biological experiments with four individual plants were collected for RNA extraction and qPCR analysis. 26S was used as an internal control. Error bars represent the SD (n = 3). Different letters a,b,c above the bars indicate a significant difference (p < 0.05), as obtained by one-way ANOVA and the LSD test.
Figure 7
Figure 7
Phenotypes of NtSOC1/NtMADS133 overexpressed transgenic lines in tobacco. (A) Growth of wild-type and two NtSOC1 overexpressed lines. Seedlings were grown in soil for 100 days. Representative plants were photographed. (B) Quantitative analysis of wild-type and two NtSOC1 overexpressed lines on the height and flower time. Three independent biological experiments with 12 individual plants per transgenic lines were calculated.

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