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作物学报 ›› 2026, Vol. 52 ›› Issue (2): 446-458.doi: 10.3724/SP.J.1006.2026.54056

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

烟草NtWRKY6基因响应ABA表达及其调控多酚合成的功能研究

詹戈锐1,2(), 余文3, 李锋1, 武明珠1, 徐馨1, 罗朝鹏1, 巫升鑫3, 杨军1, 张智强2,*(), 王中1,*()   

  1. 1中国烟草总公司郑州烟草研究院, 河南郑州 450001
    2河南农业大学烟草学院, 河南郑州 450046
    3福建省烟草专卖局烟草科究所, 福建福州 350003
  • 收稿日期:2025-04-30 接受日期:2025-10-30 出版日期:2026-02-12 网络出版日期:2025-11-12
  • 通讯作者: *张智强 E-mail:xiao_qiang8866@163.com;王中 E-mail:zhongwang1123@126.com
  • 作者简介:E-mail: zhandamei2023@163.com
  • 基金资助:
    中国烟草总公司福建省公司科技计划项目(2025350000240087)

Functional study of NtWRKY6 in response to ABA expression and regulation of polyphenol synthesis

Zhan Ge-Rui1,2(), Yu Wen3, Li Feng1, Wu Ming-Zhu1, Xu Xin1, Luo Zhao-Peng1, Wu Sheng-Xin3, Yang Jun1, Zhang Zhi-Qiang2,*(), Wang Zhong1,*()   

  1. 1Zhengzhou Tobacco Research Institute of China National Tobacco Corporation, Zhengzhou 450001, Henan, China
    2College of Tobacco Science, Henan Agricultural University, Zhengzhou 450046, Henan, China
    3Tobacco Science Research Institute of Fujian Tobacco Monopoly Bureau, Fuzhou 350003, Fujian, China
  • Received:2025-04-30 Accepted:2025-10-30 Published:2026-02-12 Published online:2025-11-12
  • Contact: *Zhang Zhi-Qiang E-mail:xiao_qiang8866@163.com;Wang Zhong E-mail:zhongwang1123@126.com
  • Supported by:
    Project of Fujian Tobacco Monopoly Bureau of China National Tobacco Corporation(2025350000240087)

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摘要：

转录因子WRKY6参与调控植物种子萌发和幼苗生长等过程, 但其是否参与调控植物多酚合成未见报道。为探究烟草WRKY6的生物学功能, 首先从栽培烟草基因组中鉴定获得一个NtWRKY6基因, 通过序列比对及进化分析发现其编码蛋白具有典型的WRKY家族功能域, 属于Group II亚型WRKY类转录因子。表达模式分析显示, NtWRKY6基因在烟草花和蒴果中表达水平较高, 且其表达受ABA、BL、JA等激素的显著诱导。过表达NtWRKY6显著增强了烟草幼苗对ABA处理的抗性, 且3个独立NtWRKY6-OE株系叶片和根中的多酚含量分别比野生型对照显著增加18.54%~77.15%与39.72%~64.07%; 相反地, ntwrky6突变体对ABA处理更加敏感, 且2个ntwrky6突变体株系叶片和根中的多酚含量分别较野生型对照降低25.05%~30.67%与19.85%~29.06%。与野生型对照相比, NtWRKY6-OE幼苗中多个多酚合成相关基因的表达水平被显著上调, 而ntwrky6幼苗中这些基因的表达水平则被显著下调。这些结果表明, NtWRKY6参与调控烟草ABA信号转导及多酚类物质的合成, 为烟草品质改良提供了新的靶标基因。

关键词: 烟草, NtWRKY6, 脱落酸, 多酚, 基因表达

Abstract:

The transcription factor WRKY6 is known to regulate seed germination and seedling growth in plants. However, its role in polyphenol biosynthesis has not yet been reported. To investigate the biological function of tobacco WRKY6, the NtWRKY6 gene was identified from the Nicotiana tabacum genome. Sequence alignment and phylogenetic analysis revealed that NtWRKY6 contains a typical WRKY domain and belongs to the Group II subfamily. NtWRKY6 was highly expressed in tobacco flowers and capsules, and its transcription was significantly induced by several plant hormones, including ABA, BL, and JA. Overexpression of NtWRKY6 significantly enhanced the resistance of tobacco seedlings to ABA treatment. In three independent NtWRKY6-overexpressing (OE) lines, polyphenol content in leaves and roots increased by 18.54%-77.15% and 39.72%-64.07%, respectively, compared to wild-type (WT) plants. In contrast, ntwrky6 mutants were more sensitive to ABA treatment, and polyphenol content in their leaves and roots decreased by 25.05%-30.67% and 19.85%-29.06%, respectively, relative to WT plants. Furthermore, the expression levels of multiple genes involved in polyphenol synthesis were significantly upregulated in NtWRKY6-OE seedlings, while they were significantly downregulated in ntwrky6 mutants. These results indicate that NtWRKY6 plays a role in regulating ABA signal transduction and polyphenol biosynthesis, providing a potential target gene for improving tobacco quality.

Key words: tobacco, NtWRKY6, abscisic acid, polyphenol, gene expression

表1

引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 引物名称
Primer name		 引物序列
Primer sequence (5'-3')
NtWRKY6-F ATGGACAAAGCTGGATGGGG qRT-NtL25-F ATTAGGCCTGATGGGACGAA
NtWRKY6-R CTGGTTCTCAAAAAGAACAGTAG qRT-NtL25-R GGCAACGTCCAAAGCATCAT
NtomWRKY6-F ATGAATATGAACCCTAGGGA qRT-RAV1-F TTGATGTTCGAAAAGGCGGT
NtomWRKY6-R GGTTCTCAAAAAGAACAGTAG qRT-RAV1-R TCACAAAACGACTCCACCCT
NsylWRKY6-F ATGGACAAAGCTGGATGGGGA CrisW6-F ATTGGATGCCAGTGGAGAAAATA
NsylWRKY6-R GGTTCTCAAAAAGAACAGTAG CrisW6-R AAACTATTTTCTCCACTGGCATC
qRT-NtWRKY6-F CAAGTGAGCACATGGACGAG
qRT-NtWRKY6-R ATGGTTGCCTCTGTTGCTTG

图1

NtWRKY6基因的鉴定及序列分析 A: 烟草WRKY6基因的克隆; 1: DNA marker; 2、3、4分别代表以栽培烟草、林烟草和绒毛状烟草cDNA为模板的扩增产物; 5、6、7代表以栽培烟草、林烟草和绒毛状烟草DNA为模板的扩增产物。B: NtWRKY6、NsylWRKY6、NtomWRKY6基因结构。C: 烟草WRKY6与同源蛋白的系统进化分析。D: 烟草WRKY6和同源蛋白的氨基酸序列比对分析; 红色方框代表典型的WRKY结构域; 三角形指示基因编辑引物对应的位置; 黑线处表示高度保守的序列区段。"

图2

NtWRKY6基因的表达模式分析 A: NtWRKY6基因在不同烟草组织器官中的相对表达水平; B: NtWRKY6基因在不同花结构中的相对表达水平; C: NtWRKY6基因在不同浓度ABA处理下的相对表达水平; D: NtWRKY6基因在不同激素处理下的相对表达水平; E: NtWRKY6基因在不同光照(0~16 h) /黑暗(16~24 h)时长下的相对表达水平; F: NtWRKY6基因在不同浓度蔗糖处理下的相对表达水平。CK: 对照; JA: 茉莉酸; GA: 赤霉素; IAA: 吲哚-3-乙酸; BL: 油菜素内酯; ABA: 脱落酸。柱形图上不同小写字母表示在0.05水平差异显著。*和**分别表示在0.05和0.01水平差异显著。"

图3

转基因材料的创制与鉴定分析 A: NtWRKY6过表达阳性植株的鉴定; B: 野生型和ntwrky6突变体株系中NtWRKY6的序列比对; C: 野生型和ntwrky6突变体株系中NtWRKY6的氨基酸序列。*和**分别表示在0.05和0.01水平差异显著。WT: 野生型; OE-#1~OE-#5表示NtWRKY6过表达株系; M1和M2分别代表2个不同的独立突变体株系。"

图4

WT、ntwrky6和NtWRKY6-OE的表型和基因表达模式分析 A: 不同处理条件下野生型、ntwrky6和NtWRKY6-OE种子的发芽率; 横坐标表示发芽天数。*和**分别表示在0.05和0.01水平差异显著。B: 不同处理条件下野生型、ntwrky6和NtWRKY6-OE幼苗的表型。C: 不同处理条件下野生型、ntwrky6和NtWRKY6-OE幼苗的根长表型。D: 不同处理条件下野生型、ntwrky6和NtWRKY6-OE幼苗的根长数值。E: 不同处理条件下野生型、ntwrky6和NtWRKY6-OE三种材料NtRAV1基因的表达水平。柱形图上不同小写字母表示差异达到显著水平(P < 0.05)。缩写同图2。"

图5

三种材料(WT、NtWRKY6-OE和ntwrky6)生化指标的测定与分析 缩写同图2。柱形图上不同小写字母表示差异达到显著水平(P < 0.05)。"

图6

NtWRKY6-OE和ntwrky6植株中的多酚含量测定 缩写同图3。柱形图上不同小写字母表示差异达到显著水平(P < 0.05)。"

图7

WT、NtWRKY6-OE和ntwrky6植株中多酚合成相关基因定量检测 柱形图上不同小写字母表示差异达到显著水平(P < 0.05)。"

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