NtWUN负调控烟草表皮毛发育

doi:10.13560/j.cnki.biotech.bull.1985.2026-0339

摘要/Abstract

摘要：

目的探究茉莉酸（jasmonic acid, JA）介导下NtWUN转录因子的作用模式，明确该转录因子调控烟草表皮毛生长发育的内在机理，为深入解析茉莉酸调控植物表皮结构发育的分子机制奠定数据与理论支撑。方法在普通烟草K326中通过同源克隆、生物信息学分析、亚细胞定位、实时荧光定量PCR等方法，解析NtWUN基因的序列特征、蛋白结构与亚细胞定位特征，明确该基因在烟草不同组织中的表达差异及在不同浓度MeJA处理条件下的表达模式；通过创制NtWUN基因过表达与基因敲除植株，开展该基因的功能验证。结果 NtWUN有2条同源序列NtWUN-1和NtWUN-2，NtWUN-1编码区全长630 bp，NtWUN-2编码区全长636 bp，分别编码209、211个氨基酸。进化树分析结果表明，NtWUN-1与林烟草高度同源，NtWUN-2与绒毛烟草高度同源，二者与枸杞中的同源基因亲缘关系较近。表达模式分析发现，NtWUN-1与NtWUN-2均在叶中表达量最高，随后依次为表皮毛、茎和花，在根中表达量最低。不同浓度MeJA处理下，NtWUN-1与NtWUN-2的相对表达量均呈先上升后下降趋势，NtWUN-1与NtWUN-2在24 h后受MeJA强烈诱导，表达量分别上调10.2倍和11.8倍。亚细胞定位结果显示，NtWUN-1与NtWUN-2都定位在细胞核。腺毛形态与密度观察发现，NtWUN过表达株系（OE8）长柄腺毛和短柄腺毛分别减少了37.5%和35.7%；敲除株系（KO2）分别增加50.0%和57.1%；上述2种转基因烟草的腺毛类型不受影响。结论 NtWUN负调控JA介导的烟草长柄腺毛和短柄腺毛的生长发育。

关键词: 烟草, NtWUN, 长柄腺毛, 短柄腺毛, 表皮毛密度, 基因编辑, 过表达, 茉莉酸

Abstract:

Objective This study aimed to elucidate the mode of action of the transcription factor NtWUN in mediating jasmonic acid (JA) signaling and to clarify the internal mechanism by which this transcription factor regulates trichome growth and development in tobacco. The findings would provide a theoretical and experimental basis for further dissecting the molecular mechanism underlying JA-mediated development of plant epidermal structures. Method In the common tobacco cultivar K326, multiple approaches including homologous cloning, bioinformatic analysis, subcellular localization assay, and quantitative real-time PCR (RT-qPCR) were adopted to characterize the sequence features and protein structure of the NtWUN gene, determine its subcellular localization properties, and clarify its tissue-specific expression patterns as well as transcriptional responses to methyl jasmonate (MeJA) treatments at varying concentrations. Furthermore, overexpression and knockout lines of NtWUN were generated to functionally validate the biological roles of this gene. Result NtWUN has two homologous sequences, NtWUN-1 and NtWUN-2. The coding region of NtWUN-1 is 630 bp in length, encoding 209 amino acids, while that of NtWUN-2 is 636 bp, encoding 211 amino acids. Phylogenetic analysis indicated that NtWUN-1 is highly homologous to Nicotiana sylvestris, and NtWUN-2 is highly homologous to N. tomentosiformis; both are closely related to homologous genes in Lycium barbarum. Expression pattern analysis showed that both NtWUN-1 and NtWUN-2 were most highly expressed in leaves, followed by trichomes, stems, and flowers, with the lowest expression in roots. Following treatment with different concentrations of MeJA, the relative expression levels of both genes initially increased and then decreased. Both genes were strongly induced by MeJA at 24 h, with expression levels upregulated by 10.2-fold and 11.8-fold, respectively. Subcellular localization results showed that NtWUN-1 and NtWUN-2 were both localized in the nucleus. Observation of trichome morphology and density revealed that the NtWUN overexpression line (OE8) showed 37.5% and 35.7% reductions in long-stalked and short-stalked glandular trichomes, respectively, while the knockout line (KO2) exhibited 50.0% and 57.1% increases in these two trichome types. The trichome types in the above transgenic lines were not affected. Conclusion The transcription factor NtWUN negatively regulates JA-mediated development of long-stalked and short-stalked glandular trichomes in tobacco.

Key words: tobacco, NtWUN, long-stalked trichome, short-stalked trichome, trichome density, gene editing, overexpression, jasmonic acid

刘娜威, 钱孟英, 张洪映, 王召军, 崔红, 闫筱筱. NtWUN负调控烟草表皮毛发育[J]. 生物技术通报, 2026, 42(9): 128-136.

LIU Na-wei, QIAN Meng-ying, ZHANG Hong-ying, WANG Zhao-jun, CUI Hong, YAN Xiao-xiao. NtWUN Negatively Regulates Tobacco Trichome Development[J]. Biotechnology Bulletin, 2026, 42(9): 128-136.

图/表 6

图1 NtWUN的克隆及生物信息学分析A：NtWUN克隆PCR电泳图；B：NtWUN及其同源蛋白系统进化树；C：NtWUN与近缘物种同源蛋白序列多重比对分析，Nt：普通烟草；Nsyl：林烟草；Ntom：绒毛烟草；Na：渐狭叶烟草；Lb：枸杞；Ca：辣椒；Sl：番茄；St：马铃薯；Gh：陆地棉；Ga：树棉

Fig. 1 Cloning and bioinformatics analysis of NtWUNA: PCR electrophoresis of NtWUN cloning. B: Phylogenetic tree of NtWUN and its homologous proteins. C: Multiple sequence alignment of NtWUN homologous proteins from closely related species. Nt: Nicotiana tabacum. Nsyl: N. sylvestris. Ntom: N. tomentosiformis. Na: N. attenuata. Lb: Lycium barbarum. Ca: Capsicum annuum. Sl: Solanum lycopersicum. St: Solanum tuberosum. Gh: Gossypium hirsutum. Ga: Gossypium arboreum

图2 NtWUN在不同组织中的表达模式*P<0.05. The same below

Fig. 2 Expression pattern of NtWUN in different tissues

图3 NtWUN响应不同浓度MeJA处理的表达分析

Fig. 3 Expression analysis of NtWUN in response to treatments with different concentrations of MeJA

图4 NtWUN蛋白的亚细胞定位

Fig. 4 Subcellular localization of the NtWUN protein

图5 NtWUN过表达植株及敲除纯合株系鉴定A：NtWUN过表达阳性株系PCR鉴定（M：DNA marker 2 000，1：ddH2O，2：NtWUN过表达质粒，3-11：9个转基因阳性单株）；B：NtWUN-2过表达阳性植株基因表达水平（**P<0.01，以K326中的表达量为参比1）；C：敲除纯合单株sgRNA区域测序分析；D：纯合突变单株中NtWUN蛋白翻译情况；红色方框标注的短横线代表蛋白翻译提前终止

Fig. 5 Identification of NtWUN-overexpressing plants and homozygous knockout linesA: PCR identification of NtWUN overexpression positive lines (M: DNA marker 2 000; 1: ddH₂O; 2: NtWUN overexpression plasmid; 3-11: nine individual transgenic positive plants). B: Gene expression levels in NtWUN-2 overexpression positive plants (**P<0.01, with the expression level in K326 as reference 1). C: Sequencing analysis of the sgRNA region in homozygous knockout plants. D: Protein translation of NtWUN in homozygous mutant plants. Short horizontal lines within red boxes indicate premature translation termination

图6 叶面表皮毛形态观察（A）与密度分析（B）同组柱形图上标有不同小字母表示0.05水平差异显著

Fig. 6 Morphological observation (A) and density analysis (B) of trichomes on the leaf surfaceDifferent lowercase letters above the bars within the same group indicate significant differences at the 0.05 level

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