Genome-wide Identification and Drought Stress Response Analysis of the NAC Gene Family in Scutellaria baicalensis

doi:10.13560/j.cnki.biotech.bull.1985.2026-0276

Abstract

Abstract:

Objective This study aimed to identify NAC gene family members in Scutellaria baicalensis Georgi and analyze their expression patterns under drought stress, providing a foundation for investigating the molecular mechanisms by which NAC transcription factors regulate baicalin biosynthesis. Method Based on S. baicalensis whole-genome data, bioinformatics analysis was performed to investigate physicochemical properties, chromosomal distribution, gene structure, conserved motifs, cis-acting elements, and collinear relationships of the SbNAC gene family. Combined with transcriptome data, key drought-responsive NAC genes were screened. Quantitative real-time PCR (RT-qPCR) was used to analyze SbNAC19, SbNAC64, and SbNAC74 tissue expression patterns in roots, stems, leaves, and flowers, as well as expression dynamics under polyethylene glycol 6000 (5% PEG) treatment and combined 5% PEG and abscisic acid (100 μmol/L ABA) treatment, and the baicalin content in each treatment group was determined using high-performance liquid chromatography (HPLC). Combined drought stress and abscisic acid treatment increased baicalin content. Result A total of 78 SbNAC genes were identified in the S. baicalensis genome. Phylogenetic analysis grouped the NAC proteins into 18 clades, with conserved structures observed across each clade, and segmental duplication was the major driver of gene family expansion. Analysis of cis-acting elements revealed that this gene family is extensively involved in regulating responses to abiotic stress and hormonal signaling. Based on transcriptomic data, three candidate genes, SbNAC19, SbNAC64, and SbNAC74, were identified as drought-responsive. Yeast one-hybrid assay results showed that all three proteins specifically bind to the CACG core sequence. PEG treatment and PEG-ABA combined treatment induced upregulation of SbNAC19 and SbNAC64 expression while downregulating SbNAC74 expression, indicating their potential roles in baicalin biosynthesis regulation. Conclusion The SbNAC family is involved in drought stress and abscisic acid signaling in S. baicalensis. Furthermore, SbNAC19, SbNAC64, and SbNAC74 may play regulatory roles in baicalin biosynthesis.

Key words: Scutellaria baicalensis Georgi, SbNAC gene family, drought stress, gene expression patterns, yeast one-hybrid assay, baicalin biosynthesis, transcriptome, bioinformatics

HAN Mei-guang, SUN Ying-xin, HAO Jin-peng, GENG Xing-yu, ZHANG Hai-hang, DI Peng, YANG Li-min, CHENG Lin. Genome-wide Identification and Drought Stress Response Analysis of the NAC Gene Family in Scutellaria baicalensis[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0276.

Figures/Tables 10

Fig. 1 Chromosomal location map of the SbNAC gene familyChr1-Chr9 represent chromosomes 1 to 9, respectively. The scale on the left indicates the physical length of chromosomes in megabase pairs (Mb). Red labels denote NAC (NAM, ATAF1/2, CUC2) family genes, and the connecting lines show the specific physical positions of these genes on the corresponding chromosomes

Fig. 2 SbNAC family collinearity analysisA: Intraspecific S. baicalensis. B: S. baicalensis versus A. thaliana and S. barbata

Fig. 3 Gene structure analysis of the SbNAC gene familyA: Conserved motif analysis. The phylogenetic tree is constructed on the left. The line length indicates the protein sequence length, and the scale bar at the bottom is in amino acids (aa). B: Conserved domain analysis. The line length corresponds to the protein sequence length, and the scale bar at the bottom is in amino acids (aa). C: Gene structure analysis. The line length corresponds to the gene sequence length, and the scale bar at the bottom is in base pairs (bp)

Fig. 4 Cis-acting elements in promoter region of the SbNAC gene family

Fig. 5 Phylogenetic relationships between S. baicalensis and A. thaliana NAC genesThe maximum likelihood method was used. The values at branch nodes represent bootstrap values (ranging from 0 to 100). Different colors in the outer ring indicate different subfamilies of the NAC family. Gene names labeled in black at the terminals of the tree represent NAC genes from A. thaliana, while those in blue represent NAC genes from S. baicalensis

Fig. 6 Baicalin content under drought and drought combined with ABA treatmentColumns represent the mean baicalin content (%) of three biological replicates. Error bars indicate standard error (SE). Different letters indicate significant differences among treatments at 0.05 level (P<0.05). The same below

Fig. 7 Correlation analysis between NAC DEGs and key differential enzymes involved in the baicalin biosynthetic pathway in S. baicalensisA: Heatmap of SbNAC DEGs. B: Protein interaction network diagram of SbNAC. C: Correlation network diagram between SbNAC DEGs and key enzymes in the baicalin synthesis pathway

Fig. 8 Tissue-specific expression pattern analysis of SbNAC19, SbNAC64, and SbNAC74 genes*, **, ***, and **** indicate significant differences at P<0.05, P<0.01, P<0.001, and P<0.000 1, respectively

Fig. 9 Differential expression of SbNAC19, SbNAC64, and SbNAC74 genes under different treatments

Fig. 10 Cis-acting element analysis and yeast one-hybrid assayA: Cis-acting elements analysis of differential genes encoding key enzymes in the baicalin synthesis pathway. B: Yeast one-hybrid assay: p53GADT7+p53His2 as positive control; pGADT7-SbNAC19, SbNAC64, and SbNAC74 + pHis2-Core as experimental group; pGADT7 + pHis2-Core as background group

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