转录因子NtMYB96a调控烟草耐旱性的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1094

摘要/Abstract

摘要：

目的基于转录组测序分析烟草NtMYB96a基因在干旱胁迫响应中的功能，解析其调控机制，为培育抗旱烟草新种质提供重要的参考基因。方法克隆NtMYB96a并构建过表达载体转化烟草，开展亚细胞定位及叶片表达模式分析；在苗期通过体内活性氧清除系统评价过表达株系的耐旱生理特性。利用RNA-seq对NtMYB96a过表达阳性植株进行转录组测序，并对差异表达基因进行GO和KEGG功能注释及富集分析。结果亚细胞定位显示NtMYB96a定位于细胞核。RT-qPCR结果表明NtMYB96a在根、茎、叶中均有表达，其中叶片表达量最高，且在不同生育时期呈现差异表达。干旱胁迫下，与野生型相比，过表达株系表现出更强的耐旱能力，表现为萎蔫程度减轻，CAT、POD和SOD活性显著提高，MDA含量显著降低。转录组分析显示，NtMYB96a通过调控光合作用、光合生物碳固定、卟啉代谢和光合天线蛋白等相关通路，及bHLH、MYB_related和WRKY等转录因子的表达参与干旱响应。结论 NtMYB96a在烟草干旱胁迫响应中发挥正调控作用，通过增强抗氧化酶活性并调控干旱相关代谢通路和转录因子网络，从而提高烟草耐旱性。

关键词: 烟草, NtMYB96a基因, 转录因子, 干旱胁迫, 表达分析, 生理特性, 转录组分析, 差异表达基因

Abstract:

Objective This study investigated the role of the tobacco NtMYB96a gene in drought-stress responses using transcriptome sequencing, with the aim of elucidating its regulatory mechanisms and providing candidate genetic resources for developing drought-resistant tobacco germplasm. Method The NtMYB96a coding sequence was cloned, and an overexpression vector was constructed and introduced into tobacco. Subcellular localization and tissue-specific expression analyses were performed. Drought-related physiological traits of NtMYB96a-overexpressing lines were evaluated at the seedling stage by assessing in vivo antioxidant activity. RNA-seq analysis was conducted on confirmed overexpression lines, and differentially expressed genes were subjected to GO and KEGG functional annotation and enrichment analyses. Result Subcellular localization showed that NtMYB96a is localized in the nucleus. RT-qPCR analysis revealed that there are expression in the roots, stems, and leaves, with the highest levels in the leaves across seedling, rosette, and vigorous growth stages. Under drought stress, NtMYB96a overexpressing lines had enhanced drought tolerance relative to wild-type plants, including reduced wilting, significantly increased CAT, POD, and SOD activities, and markedly decreased MDA content. Transcriptome analysis indicated that NtMYB96a alters the expression of genes involved in photosynthesis, carbon fixation, porphyrin metabolism, and photosynthetic antenna proteins, as well as several drought-responsive transcription factor families such as bHLH, MYB-related, and WRKY. Conclusion NtMYB96a positively regulates tobacco drought-stress responses by enhancing antioxidant enzyme activity and modulating key drought-related metabolic pathways and transcription factor networks, thereby improving plant drought tolerance.

Key words: tobacco, NtMYB96a gene, transcription factors, drought stress, expression analysis, physiological characteristics, transcriptome analysis, differentially expressed genes

刘青媛, 吴洪启, 陈秀娥, 陈剑, 姜远泽, 何燕子, 喻奇伟, 刘仁祥. 转录因子NtMYB96a调控烟草耐旱性的功能研究[J]. 生物技术通报, 2026, 42(4): 239-250.

LIU Qing-yuan, WU Hong-qi, CHEN Xiu-e, CHEN Jian, JIANG Yuan-ze, HE Yan-zi, YU Qi-wei, LIU Ren-xiang. Function of Transcription Factor NtMYB96a in Regulating the Tolerance of Tobacco to Drought[J]. Biotechnology Bulletin, 2026, 42(4): 239-250.

图/表 9

图1 AtMYB96与NtMYB96s的氨基酸序列比对

Fig. 1 Amino acid sequence alignment of AtMYB96 and NtMYB96s

图2 NtMYB96a保守结构域分析

Fig. 2 Analysis of the conserved domains in NtMYB96a

图3 NtMYB96a基因的筛选及其表达特性分析A：干旱胁迫转录组数据中NtMYB96s基因的FPKM值；B：干旱胁迫下NtMYB96a和NtMYB96b基因表达情况；C：NtMYB96a在烟草苗期组织及不同发育时期叶片中的表达水平分析；D：NtMYB96a的亚细胞定位分析。不同小写字母表示在P<0.05水平差异显著（t检验），数据均为3次生物学重复的平均值±标准差，下同

Fig. 3 Screening and expression characterization analysis of the NtMYB96a geneA: FPKM values of NtMYB96s genes from transcriptome data under drought stress. B: Expressions of NtMYB96a and NtMYB96bunder drought stress. C: Expression analysis of NtMYB96a in tissues of tobacco seedlings and in leaves at different developmental stages. D: Subcellular localization analysis of NtMYB96a. Different lowercase letters indicate statistically significant differences at P<0.05 (t test). All data are presented as mean ± SD from three biological replicates. The same below

图4 NtMYB96a基因的克隆及其过表达株系的创制A：NtMYB96a基因的CDS扩增；B：NtMYB96a过表达烟草阳性植株鉴定（M：marker；-：阴性对照；+：阳性对照；1‒6：T0代阳性植株）；C：NtMYB96a在过表达植株中的表达水平分析；星号表示与野生型（WT）植株的差异显著性，t检验，3次生物学重复，* P<0.05，** P<0.01，下同

Fig. 4 Cloning and generation of overexpressing lines of NtMYB96aA: Amplification of the CDS region of NtMYB96a gene. B: Identification of positive transgenic tobacco plants overexpressing NtMYB96a (M: marker; -: negative control; +: positive control; 1-6: T0 generation positive plants). C: Expression analysis of NtMYB96a in overexpression plants. Asterisks indicate statistically significant differences compared with the wild-type (WT) plants. t test, three biological replicates, * P<0.05, ** P<0.01. The same below

图5 干旱胁迫下OE-NtMYB96a#1植株的苗期生长状况和抗氧化物酶活性和丙二醛含量的测定A：干旱胁迫处理下植株的生长表型（标尺=10 cm）；B‒E：干旱胁迫下抗氧化物酶活性和丙二醛含量分析

Fig. 5 Determination of seedling growth, antioxidant enzyme activity, and MDA content ofOE-NtMYB96a#1plants under drought stressA: Growing phenotype of plants under drought stress treatment (scale bar=10 cm). B-E: Analysis of antioxidant enzyme activity and MDA content under drought stress

图6 过表达NtMYB96a基因对烟草干旱胁迫转录组的影响A：差异表达基因数量；B：差异表达基因韦恩图；图A和B中4个组分别是干旱处理下0 h的WT（WT_0 h）和OE-NtMYB96a#1（OE_0 h），干旱处理下24 h的WT（WT_24 h）和OE-NtMYB96a#1（OE_24 h）；C：OE-NtMYB96a#1特有DEGs的GO富集分析；CC：细胞组分；BP：生物过程；D：OE-NtMYB96a#1特有DEGs的KEGG富集分析

Fig. 6 Effects of NtMYB96a overexpression on the transcriptome of tobacco under drought stressA: Number of differentially expressed genes(DEGs). B: Venn diagram of DEGs. In panel A and B, the four groups are: WT under drought treatment at 0 h (WT_0 h), OE-NtMYB96a#1 under drought treatment at 0 h (OE_0 h), WT under drought treatment at 24 h (WT_24 h) , and OE-NtMYB96a#1 under drought treatment at 24 h (OE_24 h) . C: GO enrichment analysis of OE-NtMYB96a#1-specific DEGs. CC: Cellular component. BP: Biological process. D: KEGG enrichment analysis of DEGs unique to OE-NtMYB96a#1

图7 干旱胁迫下过表达NtMYB96a特有DEGs的热图分析A：光合作用；B：光合生物的碳固定；C：卟啉代谢；D：光合作用‒天线蛋白

Fig. 7 Heatmap analysis of DEGsspecifically regulated by NtMYB96a overexpressionunder drought stressA: Photosynthesis. B: Carbon fixation of photosynthetic organisms. C: Porphyrin metabolism. D: Photosynthesis-antenna protein

图8 干旱胁迫下过表达NtMYB96a特有DEGs中转录因子家族分析A：转录因子家族统计；B：干旱胁迫下转录因子家族成员的表达分析

Fig. 8 Analysis of transcription factor families among DEGs specifically regulated by NtMYB96a overexpression under drought stressA: Statistics of transcription factor families. B: Expression analysis of transcription factor family members under drought stress

图9 干旱胁迫下WT和过表达NtMYB96a中叶绿素合成及光合作用相关基因的表达水平

Fig. 9 Expressions of genes related to chlorophyll biosynthesis and photosynthesis in wild-type and NtMYB96a -overexpressing plants under drought stress

参考文献 32

[1]	彭文婷, 李庆, 代明球. 作物抗旱关键调控因子的挖掘与利用 [J]. 科学通报, 2025, 70(19): 3149-3167.
	Peng WT, Li Q, Dai MQ. Exploitation of key regulatory factors and breeding strategies for crop drought resistance [J]. Chin Sci Bull, 2025, 70(19): 3149-3167.
[2]	李佳, 刘涛, 马菊莲, 等. 烟草响应干旱胁迫与抗旱遗传育种研究进展 [J]. 江苏农业科学, 2023, 51(8): 34-43.
	Li J, Liu T, Ma JL, et al. Research progress on drought stress response and drought resistance genetic breeding of tobacco [J]. Jiangsu Agric Sci, 2023, 51(8): 34-43.
[3]	Zhang DW, Zhou HP, Zhang Y, et al. Diverse roles of MYB transcription factors in plants [J]. J Integr Plant Biol, 2025, 67(3): 539-562.
[4]	Liu YH, Shen Y, Liang M, et al. Identification of peanut AhMYB44 transcription factors and their multiple roles in drought stress responses [J]. Plants, 2022, 11(24): 3522.
[5]	Zhao HY, Zhao HQ, Hu YF, et al. Expression of the sweet potato MYB transcription factor IbMYB48 confers salt and drought tolerance in Arabidopsis [J]. Genes, 2022, 13(10): 1883.
[6]	Gao J, Zhao Y, Zhao ZK, et al. RRS1 shapes robust root system to enhance drought resistance in rice [J]. New Phytol, 2023, 238(3): 1146-1162.
[7]	Pil Joon Seo SBL. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis [J]. Plant Cell, 2011, 23(3): 1138-1152.
[8]	李霞, 张泽伟, 刘泽军, 等. 马铃薯转录因子StMYB96的克隆及功能研究 [J]. 生物技术通报, 2025, 41(7): 181-192.
	Li X, Zhang ZW, Liu ZJ, et al. Cloning and functional study of transcription factor StMYB96 in potato [J]. Biotechnol Bull, 2025, 41(7): 181-192.
[9]	He JJ, Li CZ, Hu N, et al. ECERIFERUM1-6A is required for the synthesis of cuticular wax alkanes and promotes drought tolerance in wheat [J]. Plant Physiol, 2022, 190(3): 1640-1657.
[10]	赵先炎, 齐晨辉, 姜翰, 等. 苹果MpMYB96基因克隆和功能鉴定 [J]. 植物生理学报, 2019, 55(6): 783-792.
	Zhao XY, Qi CH, Jiang H, et al. Cloning and functional identification of MpMYB96 gene in Malus pumila [J]. Plant Physiol J, 2019, 55(6): 783-792.
[11]	赵海英, 刘致远, 曹际玲. 纳米银对不同品种玉米幼苗生长和生理特性的影响 [J]. 热带农业科学, 2025, 45(7): 50-57.
	Zhao HY, Liu ZY, Cao JL. Effects of silver nanoparticles on the growth and physiological characteristics of different varieties of maize seedlings [J]. Chin J Trop Agric, 2025, 45(7): 50-57.
[12]	孙美华, 孙慧贤, 赵晏俪, 等. 不同供氮水平对番茄果实心室数量的影响 [J]. 园艺学报, 2025, 52(10): 2737-2747.
	Sun MH, Sun HX, Zhao YL, et al. Effects of different nitrogen concentration on the locule number in tomato fruit [J]. Acta Hortic Sin, 2025, 52(10): 2737-2747.
[13]	Kim JB, Kim SH, Bae DH. The impacts of global warming on arid climate and drought features [J]. Theor Appl Climatol, 2023, 152(1): 693-708.
[14]	王亚虹, 许自成, 高森, 等. 烟草干旱胁迫研究进展 [J]. 节水灌溉, 2016(12): 103-107, 111.
	Wang YH, Xu ZC, Gao S, et al. Research progress of tobacco drought stress [J]. Water Sav Irrig, 2016(12): 103-107, 111.
[15]	金伊楠, 张豪洋, 张璐翔, 等. 烟草抗旱相关基因的研究进展 [J]. 烟草科技, 2020, 53(3): 18-26.
	Jin YN, Zhang HY, Zhang LX, et al. Research progress of drought-resistance related genes in tobacco [J]. Tob Sci Technol, 2020, 53(3): 18-26.
[16]	Wei QH, Zhang F, Sun FS, et al. A wheat MYB transcriptional repressor TaMyb1D regulates phenylpropanoid metabolism and enhances tolerance to drought and oxidative stresses in transgenic tobacco plants [J]. Plant Sci, 2017, 265: 112-123.
[17]	Liu ZY, Li JN, Li S, et al. The 1R-MYB transcription factor SlMYB1L modulates drought tolerance via an ABA-dependent pathway in tomato [J]. Plant Physiol Biochem, 2025, 222: 109721.
[18]	Yan ML, Li XX, Ji XY, et al. An R2R3-MYB transcription factor PdbMYB6 enhances drought tolerance by mediating reactive oxygen species scavenging, osmotic balance, and stomatal opening [J]. Plant Physiol Biochem, 2025, 220: 109536.
[19]	Zhang XB, Lei L, Lai JS, et al. Effects of drought stress and water recovery on physiological responses and gene expression in maize seedlings [J]. BMC Plant Biol, 2018, 18(1): 68.
[20]	Wang YX, Zhang MH, Li XY, et al. Overexpression of the wheat TaPsb28 gene enhances drought tolerance in transgenic Arabidopsis [J]. Int J Mol Sci, 2023, 24(6): 5226.
[21]	Wang LX, Wang BW, Du QZ, et al. Allelic variation in PtoPsbW associated with photosynthesis, growth, and wood properties in Populus tomentosa [J]. Mol Genet Genomics, 2017, 292(1): 77-91.
[22]	Cui L, Zheng FY, Zhang DD, et al. Tomato methionine sulfoxide reductase B2 functions in drought tolerance by promoting ROS scavenging and chlorophyll accumulation through interaction with Catalase 2 and RBCS3B [J]. Plant Sci, 2022, 318: 111206.
[23]	Liu EL, Xu LL, Luo ZQ, et al. Transcriptomic analysis reveals mechanisms for the different drought tolerance of sweet potatoes [J]. Front Plant Sci, 2023, 14: 1136709.
[24]	Karami S, Shiran B, Ravash R. Molecular investigation of how drought stress affects chlorophyll metabolism and photosynthesis in leaves of C₃ and C₄ plant species: a transcriptome meta-analysis [J]. Heliyon, 2025, 11(3): e42368.
[25]	Wei GQ, Zhang YT, Yang Y, et al. CfCHLM, from Cryptomeria fortunei, promotes chlorophyll synthesis and improves tolerance to abiotic stresses in transgenic Arabidopsis thaliana [J]. Forests, 2024, 15(4): 628.
[26]	Ye JJ, Lin XY, Yang ZX, et al. The light-harvesting chlorophyll a/b-binding proteins of photosystem Ⅱ family members are responsible for temperature sensitivity and leaf color phenotype in albino tea plant [J]. J Adv Res, 2024, 66: 87-104.
[27]	Cheng JQ, Wu TT, Zhou Y, et al. The alternative splicing of HvLHCA4.2 enhances drought tolerance in barley by regulating ROS scavenging and stomatal closure [J]. Int J Biol Macromol, 2025, 307: 142384.
[28]	Zhang HY, Wang YL, Song XY, et al. BcLhcb2.1, a Light-harvesting chlorophyll a/b-binding protein from Wucai, plays a positive regulatory role in the response to abiotic stress [J]. Sci Hortic, 2025, 339: 113759.
[29]	Zhao S, Gao HB, Luo JW, et al. Genome-wide analysis of the light-harvesting chlorophyll a/b-binding gene family in apple (Malus domestica) and functional characterization of MdLhcb4.3, which confers tolerance to drought and osmotic stress [J]. Plant Physiol Biochem, 2020, 154: 517-529.
[30]	Liang YF, Ma F, Li BY, et al. A bHLH transcription factor, SlbHLH96, promotes drought tolerance in tomato [J]. Hortic Res, 2022, 9: uhac198.
[31]	Zhao PC, Hou SL, Guo XF, et al. A MYB-related transcription factor from sheepgrass, LcMYB2, promotes seed germination and root growth under drought stress [J]. BMC Plant Biol, 2019, 19(1): 564.
[32]	Gulzar F, Fu JY, Zhu CY, et al. Maize WRKY transcription factor ZmWRKY79 positively regulates drought tolerance through elevating ABA biosynthesis [J]. Int J Mol Sci, 2021, 22(18): 10080.