Cloning and Functional Study of Transcription Factor StMYB96 in Potato

doi:10.13560/j.cnki.biotech.bull.1985.2025-0105

Abstract

Abstract:

Objective MYB transcription factors (TFs) play critical roles in plant responses to abiotic stress by regulating the expressions of stress response genes. This study is aimed to investigate the role of StMYB96 in responses to abiotic stress in potato. Method The StMYB96 gene was cloned and subjected to bioinformatics analysis, subcellular localization, and expression pattern analysis. By expressing StMYB96 in vitro and combining it with DNA affinity purification sequencing (DAP-seq), the target genes of StMYB96 and the abiotic stress-responding pathways in which they participate were identified genome-wide. Result The length of open reading frame (ORE) of StMYB96 was 1 002 bp, it encoded a peptide of 333 amino acids. Multiple sequence alignment revealed that StMYB96 was closely related to SlMYB306 (Solanum lycopersicum), CaMYB306 (Capsicum annuum), and LbMYB306 (Lycium barbarum). Subcellular localization analysis indicated that StMYB96 was localized in the nucleus. The quantitative reverse transcription-PCR (RT-qPCR) analysis showed that StMYB96 was expressed in the leaves, stems, and roots, with the highest expression observed in the stems. The expression of StMYB96 was significantly up-regulated under drought stress but down-regulated under low-temperature treatment. DAP-seq analysis revealed that StMYB96 mainly bound to three specific binding sites in the promoters of 8 837 target genes, which were involved in various metabolic pathways. Notably, genes associated with the flavonoid biosynthesis pathway and the fatty acid elongation pathway were directly regulated by StMYB96. Conclusion StMYB96 may play a role in responding to drought and low-temperature stresses by regulating different metabolic pathways.

Key words: potato, transcription factors, abiotic stress, DAP-seq, StMYB96

LI Xia, ZHANG Ze-wei, LIU Ze-jun, WANG Nan, GUO Jiang-bo, XIN Cui-hua, ZHANG Tong, JIAN Lei. Cloning and Functional Study of Transcription Factor StMYB96 in Potato[J]. Biotechnology Bulletin, 2025, 41(7): 181-192.

Figures/Tables 11

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5'-3'）	用途 Usage
StMYB96-F	CGACGACAAGACCGTGACCATGGGAAGACCACCTTGCTG	基因克隆
StMYB96-R	GAGGAGAAGAGCCGTCGAAAAAAGTCAGCAGATTCAC	Gene cloning
StMYB96-qRT-F	TCAAAGGGACAGTGGGAGAG	荧光定量PCR
StMYB96-qRT-R	AGAGCTGGATCCTGTAACGG	RT-qPCR
StEF1α-qRT-F	GACAAGCGTGTTATTGAGAGG	内参基因
StEF1α-qRT-R	CACAGTGCAGTAGTACTTAGTG	Reference gene
StMYB96-OX-F	CACGGGGGACTCTAGAATGGGAAGACCACCTTGCTG	过表达载体的构建
StMYB96-OX-R	GGGGAAATTCGAGCTCTCAAAAAAAGTCAGCAGATT	Construction of overexpression vector

Fig. 1 Cloning and sequence analysis of StMYB96 in S. tuberosum L.A: ‍Electrophoretogram of PCR product of StMYB96 gene in S. tuberosum L. (M: DNA marker; P: StMYB96 PCR amplified product). B: Gene structure of StMYB96. C: ‍Conserved domain analysis ofStMYB96 protein. D: StMYB96 gene sequence and deduced amino acid sequence (Green marked nucleotide sequence and pink marked amino acid sequence indicate MYB domain; the asterisk (*) refers to stop codon)

Fig. 2 Prediction of secondary and tertiary structure of StMYB96 proteinA: The secondary structure prediction of StMYB96 protein. Capital letters indicate amino acid sequence of StMYB96 protein. Lowercase letters indicate different secondary structures, where c, h, e and t indicate random coil, α-helix, extended strand and β-turn, respectively. B: The tertiary structure prediction of StMYB96 protein

Fig. 3 Multiple sequence alignment between StMYB96 and its homologous proteins from other speciesDark blue indicates amino acids completely conserved, pink indicates amino acids partially conserved, and light blue indicates amino acids similar

Fig. 4 Phylogenetic tree of StMYB96 proteinStMYB96: Solanum tuberosum (PGSC0003DMG400019535); SlMYB306: Solanum lycopersicum (XP_004236011.1); CaMYB306: Capsicum annuum (XP_016563223.1); LbMYB306b: Lycium barbarum (XP_060205081.1); NtMYB306: Nicotiana tabacum (XP_016452464.1); ArMYB96: Actinidia rufa (GFZ17560.1); AtMYB96: Arabidopsis thaliana (NP_201053.2); AtMYB94 (OAP04445.1)

Fig. 5 Subcellular localization of StMYB96 in S. tuberosum L.A: The structure of 35S::StMYB96::GFP vector. B: Subcellular localization of GFP protein and StMYB96::GFP fusion protein in Nicotiana benthamiana epidermal cells. (From left to right: GFP fluorescence, chloroplast self-luminescence, bright-field, and merged microscope image; Scale bar = 50 µm)

Fig. 6 Expression analysis of StMYB96 in various tissues and in response to various abiotic stressesA: Relative expressions of StMYB96 in different tissues. B: Expression proﬁles of StMYB96 in response to drought stress. C: Expression proﬁles of StMYB96 in response to low temperature stress. HT: Hours of treatment. ** indicates signiﬁcant difference at P<0.01

Fig. 7 Genome-wide identification of direct binding sites of StMYB96A: Venn diagram of peak number specifically binding to genomic DNA by StMYB96 identified in two biological replicates. B: Distribution of StMYB96 binding sites related to transcription start sites (TSS ±2 000 bp). C: Distribution of StMYB96 binding sites in potato genome. D: Binding motif of StMYB96

Fig. 8 KEGG pathway enrichment analysis and GO functional annotation analysis of StMYB96 target genesA: KEGG pathway enrichment analysis of StMYB96 target genes. The x-axis refers to the Rich factor corresponding to the pathways, the y-axis refers to the pathway names, the sizes of the dots indicate the number of target genes in each pathway, and the color of the dot indicates the size of the P-value. B: GO functional annotation analysis of StMYB96 target genes. The x-axis refers to the next-level GO terms under the three main GO categories (biological process, molecular function, and cellular component), and the y-axis refers to the proportion of target genes annotated to each term

Fig. 9 StMYB96-target genes involved in the responses to drought and low-temperature stressA: StMYB96-target genes involved in the flavonoid biosynthesis and fatty acid elongation pathway. B: StMYB96-target genes involved in the CBF signaling pathway. C: StMYB96-target genes involved in some functional annotations and drought stress response. Rep1 and Rep2 refer to two biological replicates. Fold enrichment indicates the binding enrichment level of StMYB96 in the promoter regions of genes

Fig. 10 A working model hypothesis of StMYB96 in response to drought and low-temperature stress in potatoSolid lines indicate known or experimentally validated regulatory pathways, dashed lines indicate hypothesized regulatory pathways, arrows indicate activation, and T-shaped arrows indicate inhibition

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