Comprehensive analysis of the NAC transcription factor gene family in Sophora tonkinensis Gagnep

Abstract

Background: Sophora tonkinensis Gagnep. has long been utilized in the treatment of anti-inflammatory and pain-relieving, with its principal active compounds being alkaloids and flavonoids. NAC transcription factors, a large family of plant-specific regulators, play pivotal roles in growth, development, stress responses, and secondary metabolism. However, comprehensive genome-wide characterization of S. tonkinensis NAC gene family (StNAC) remains unexplored.

Results: This study identified 85 NAC proteins from the S. tonkinensis genome database. Phylogenetic analysis revealed that StNAC proteins were categorized into 15 subgroups based on their homology with Arabidopsis thaliana NAC proteins. Gene structure analysis demonstrated a variation in intron numbers ranging from 1 to 7, with a majority of StNAC genes containing 2-3 introns. Chromosomal distribution analysis indicated an uneven spread of StNAC genes across 9 chromosomes, with the highest number of StNAC genes on Chr3. Detection of 4 tandem duplicates and 32 segmental duplicates revealed that segmental duplication primarily drive StNAC genes amplification. Prediction of cis-regulatory elements suggested the involvement of StNAC genes in growth, stress responses, and hormone regulation. Gene expression analysis showed substantial variability expression of StNAC genes across different tissues. Notably, eight StNAC genes were identified as significantly associated alkaloid and flavonoid levels. qRT-PCR validation indicated that five genes were highly expressed in tissues, corroborating transcriptome data.

Conclusion: These findings offer valuable insights for further functional characterization of NAC genes and their potential roles in alkaloid and flavonoid biosynthesis in S. tonkinensis.

Keywords: Sophora tonkinensis Gagnep; Alkaloid; Flavonoid; Gene family; NAC transcription factor.

Fig. 1

Chromosomal location of the NAC family genes in S. tonkinensis. The vertical lines represent the chromosomes of S. tonkinensis. Chromosome numbering was located at the top of the chromosome. The scale on the left stands for the length of the chromosome. Tandem duplicated genes are highlighted in red

Fig. 2

Phylogenetic relationship among NAC superfamily genes of S. tonkinensis and A. thaliana. Construction of a rootless phylogenetic tree with maximum likelihood (ML) method These proteins were divided into 15 subgroups and each of them was assigned a different color. The numerical values on the phylogenetic tree branches represent bootstrap support percentages, with higher values indicating stronger statistical confidence in the corresponding clade topology. The Red circles indicated NAC genes in A. thaliana, and green pentagrams showed NAC genes in S. tonkinensis

Fig. 3

Analysis of motifs and gene structure of StNAC. (A) Phylogenetic tree of StNAC gene family members. (B) Conserved motifs of StNAC genes. each motif was depicted by a colored box, the position of each motif can be estimated using the scale at the bottom, and different subclades of the rootless tree were marked with different colors. (C) Predicted gene structure of StNAC genes. CDSs, untranslated regions (UTRs), and introns were shown using blue boxes, green bars, and grey lines

Fig. 4

Cis-elements analysis of NAC genes in S. tonkinensis. The color of the box from yellow to green signifies the number of cis-acting elements, and the different colored modules on the left side denote the different classes of cis-acting elements

Fig. 5

Collinearity analysis of the NAC gene family in S. tonkinensis. Gray lines indicate all homozygous blocks in the genome. Segmental repeats of NAC gene pairs on different stainings were indicated and labeled with different color colors

Fig. 6

Collinearity analysis of the NAC gene family between S. tonkinensis (StChr1-StChr9) and A. thaliana (AtChr1-AtChr5). Gray lines indicate co-lined blocks within the S. tonkinensis and A. thaliana genomes, and red lines highlight co-lined NAC gene pairs between the S. tonkinensis and A. thaliana

Fig. 7

Expression levels of StNAC genes in roots, stems, leaves, and seeds using RNA-seq. Red to blue color represents gene expression from high to low. Values of 1.5 and − 1.5 represents relative expression values

Fig. 8

Correlation analysis between StNAC genes and principal constituents content was analyzed by Spearman rank correlation analysis in R (v3.6.2), and p < 0.05 (*) was considered statistically significant

Fig. 9

Expression of eight StNAC genes in various tissues of S. tonkinensis. The mean ± SD of three independent repetitions is indicated by the error bars. Different lowercase letters (a, b, c, and d) denote significant differences (p < 0.05)