Function of Transcription Factor NtMYB96a in Regulating the Tolerance of Tobacco to Drought

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

Abstract

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

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.

Figures/Tables 9

Fig. 1 Amino acid sequence alignment of AtMYB96 and NtMYB96s

Fig. 2 Analysis of the conserved domains in NtMYB96a

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

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

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

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

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

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

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

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