黄芩NAC基因家族鉴定及干旱胁迫响应分析

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

摘要/Abstract

摘要：

目的挖掘黄芩SbNAC基因家族成员，分析其在干旱胁迫下的表达特征，为探究NAC转录因子调控黄芩苷生物合成的分子机制奠定基础。方法基于黄芩的全基因组数据，采用生物信息学方法对SbNAC基因家族的理化性质、染色体定位、基因结构、保守基序、顺式作用元件及共线性关系进行系统分析。结合转录组数据筛选响应干旱胁迫的关键NAC基因，利用实时荧光定量PCR技术分析SbNAC19、SbNAC64和SbNAC74在黄芩根、茎、叶、花中的表达模式，以及干旱（5% PEG）和干旱联合脱落酸（5% PEG+100 μmol/L ABA）处理下不同时间点的表达动态。利用高效液相色谱法检测各处理下的黄芩苷含量。结果从黄芩基因组中共鉴定出78个SbNAC基因，系统进化分析将NAC蛋白分为18个分支且各分支的结构相对保守，片段重复是基因扩增的主要驱动力，顺式作用元件分析发现该家族广泛参与非生物逆境胁迫及激素信号的响应调控。干旱联合脱落酸处理可提高黄芩苷含量。根据转录组数据筛选获得3个响应干旱胁迫的候选基因SbNAC19、SbNAC64和SbNAC74，酵母单杂交结果显示这3个基因均能特异性结合CACG核心序列，PEG及PEG+ABA处理诱导SbNAC19和SbNAC64表达上调，而SbNAC74表达下调，揭示其可能参与调控黄芩苷的生物合成。结论 SbNAC家族参与黄芩干旱胁迫及ABA信号响应过程，SbNAC19、SbNAC64和SbNAC74对黄芩苷合成具有潜在调控作用。

关键词: 黄芩, SbNAC基因家族, 干旱胁迫, 基因表达模式, 酵母单杂交, 黄芩苷合成, 转录组, 生物信息学分析

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

韩美光, 孙英新, 郝金鹏, 耿兴雨, 张海航, 邸鹏, 杨利民, 程林. 黄芩NAC基因家族鉴定及干旱胁迫响应分析[J]. 生物技术通报, 2026, 42(11): 196-209.

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, 2026, 42(11): 196-209.

图/表 10

图1 SbNAC基因家族的染色体定位图谱Chr1-Chr9为1-9号染色体。左侧刻度为染色体长度（Mb），红色标注为NAC家族基因，引线指示基因在染色体上的具体位置

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 family genes, and the connecting lines show the specific physical positions of these genes on the corresponding chromosomes

图2 SbNAC家族共线性分析A：黄芩种内；B：黄芩与拟南芥、半枝莲

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

图3 SbNAC基因家族的基因结构分析A：保守基序分析，左侧为系统发育树，线条长度表示蛋白序列长度，底部标尺单位为氨基酸（aa）；B：保守结构域分析，线条长度对应蛋白序列长度，底部标尺单位为氨基酸（aa）；C：基因结构分析，线条长度对应基因序列长度，底部标尺单位为碱基对（bp）

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)

图4 SbNAC基因家族启动子区域顺式作用元件

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

图5 黄芩和拟南芥NAC基因的系统发育关系采用最大似然法，分支节点数值为自展支持值（取值0-100），外圈不同颜色代表NAC家族的不同亚家族，进化树末端黑色字体为拟南芥NAC基因名称、蓝色字体为黄芩NAC基因名称

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

图6 干旱及干旱联合ABA处理下黄芩苷含量柱形代表3次生物学重复的黄芩苷含量平均值（%），误差线表示标准误（SE），不同字母表示处理间在0.05水平上差异显著（P<0.05）。下同

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

图7 黄芩NAC差异基因与黄芩苷通路差异关键酶间的相关性分析A：SbNAC差异基因热图；B：SbNAC差异蛋白互作网络图；C：SbNAC差异基因和黄芩苷合成通路关键酶差异基因相关性网络图

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

图8 SbNAC19、SbNAC64和SbNAC74基因组织表达特异性分析*， **， ***， **** 分别表示差异显著水平为P<0.05、P<0.01、P<0.001和P<0.000 1

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

图9 不同处理下SbNAC19、SbNAC64和SbNAC74基因的表达差异

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

图10 顺式作用元件分析和酵母单杂交分析A：编码黄芩苷合成通路关键酶的差异基因顺式作用元件分析；B：酵母单杂交实验；p53GADT7+p53His2作为阳性对照，pGADT7-SbNAC19、SbNAC64和SbNAC74+ pHis2-Core作为实验组，pGADT7+pHis2-Core作为背景组

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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