Analysis of Expression Characteristics and Identification of Interaction Proteins of BnaABF2 Transcription Factor in Brassica napus

doi:10.11983/CBB24019

Abstract

Abstract: ABF transcription factors are collectively referred to as basic leucine zipper proteins that can specifically recognize and bind to ABA-responsive elements (ABRE), participating in ABA signal transduction and serving as regulators of ABA signal transcriptional responses. This study analyzed the protein encoded by the BnaABF2 gene in Brassica napus. Subcellular localization results showed that the BnaABF2 protein is localized in the nucleus. Analysis of transcriptional activity in the yeast system indicated that BnaABF2 has no transcriptional activation activity; qRT-PCR detection revealed that the expression level of BnaABF2 is highest in leaves. We also found that ABA treatment, simulated drought, and salt stress can induce the expression of BnaABF2; BiFC results showed that BnaMPK1/2/6/7/9/12/13 can interact with BnaABF2. Dual-LUC results suggested that BnaMPK7 may enhance the transcriptional regulation of BnaABF2 on downstream target genes through phosphorylation. This study initially explored the basic characteristics and interacting proteins of the transcription factor BnaABF2, providing theoretical guidance for understanding its functions and mechanisms.

Key words: Brassica napus, ABF2, subcellular localization, MPK, interaction proteins

Liuqing Yang, Jin Wang, Jingli Yan, Qinqin Chen, Haokun Cheng, Chun Li, Peiyu Zhao, Bo Yang, Yuanqing Jiang. Analysis of Expression Characteristics and Identification of Interaction Proteins of BnaABF2 Transcription Factor in Brassica napus[J]. Chinese Bulletin of Botany, 2025, 60(1): 49-61.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24019

https://www.chinbullbotany.com/EN/Y2025/V60/I1/49

Figures/Tables 8

Figure 1 Cis-elements of two BnaABF2 promoters ABRE: Abscisic acid responsiveness element; ARE: Anaerobic induction element; Light: Light responsiveness element; TCA- element: Salicylic acid responsiveness element; TC-rich repeats: Defense and stress responsiveness element; TGA-element: Auxin responsiveness element; CGTCA-motif: MeJA-responsiveness motif; TGACG-motif: Jasmonic acid responsiveness motif; Circadian: Circadian rhythm

Figure 2 Sequence alignment of conserved domains of BnaABF2 with ABF from other species Left: Protein conserved motifs of ABF2/3 alignment; Middle: ABF2/3 protein conserved domain alignment; Right: Transcript structure alignment of coding region (CDS) and untranslated region (UTR) of ABF2/3.

Figure 3 A phylogenetic tree of BnaABF2 and ABF proteins from other species The branches of the evolutionary tree showed genetic distance with a confidence level of 100.

Figure 4 Subcellular localization of BnaABF2 pYJHF-BnaABF2 or pYJHF (control) plasmid were infiltrated into tobacco leaves, and fluorescence signals were observed two days later. GFP: Green fluorescent protein signal; NLS-mCherry: Nuclear localization marker; Bright: Bright field; Merge: Superimposed field. Bars=50 μm

Figure 5 Analysis of transcriptional activity of BnaABF2 in yeast system BD (negative control), BD-BnaABF2, BD-BnaMYB111 (positive control), BD-BnaABF2 5m, BD-BnaABF2 5D and BD-BnaABF2 Co were transferred into yeast for titration experiment. The dilution gradients of yeast cells were marked with black triangles.

Figure 6 Expression patterns of BnaABF2 gene in different tissues and under different stresses and plant growth regulator treatments (A) The expression of BnaABF2 in different tissues; (B) The expression of BnaABF2 under various stresses and plant growth regulator treatments, the treatments included 50 μmol·L-1 ABA, 15% PEG8000, cold (4°C), heat (38°C) and salinity (200 mmol·L-1 NaCl). Values are means±SE. The significant differences in t-test were indicated by asterisk, * P<0.05; ** P<0.01

Figure 7 Screening of interaction between BnaABF2 and BnaMPK proteins through bimolecular fluorescence complementation assay The plasmids of BnaABF2-YFPc and YFPn-BnaMPK1/2/6/ 7/9/12/13 and NLS-mCherry were co-transfected into tobacco (28 d). Four days later, the fluorescence signal was observed. YFPn: Yellow fluorescent protein N-terminal sequence; YFPc: Yellow fluorescent protein C-terminal sequence; YFP: Yellow fluorescent protein signal; NLS-mCherry: Nuclear localization red fluorescence signal; Bright: Bright field; Merge: Superimposed field. Bars=50 μm

Figure 8 Dual-luciferase reporter system detects the effect of BnaABF2 on the transcriptional activity of AtRD29B promoter under abscisic acid (ABA) treatment (A) A schematic diagram of the reporter and effector; (B) The LUC/REN ratio is used to indicate the ability of the effector to activate the reporter (dpi: Days post-infiltration). Different lowercase letters indicate significant differences between different treatments (P<0.05).

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