Cloning and Expression Analysis of the UV-B Receptor Gene McUVR8 in Mentha canadensis L.

doi:10.13560/j.cnki.biotech.bull.1985.2025-0013

Abstract

Abstract:

Objective The ultraviolet-B (UV-B) receptor UVR8 plays a key role in regulating plant growth and development in response to adversity stress. The aim is to explore the protein properties and expression characteristics of McUVR8, a UV-B receptor gene in mint (Mentha canadensis L.), and to predict its interacting proteins, thus to provide theoretical references for the subsequent probing of the function of McUVR8. Method The McUVR8 gene was obtained by RT-PCR, and its physicochemical properties, protein structure and subcellular localization, and interacting proteins were analyzed and predicted using a variety of bioinformatics tools. Tissue and leaf sequence expression patterns of McUVR8 and its expression changes under different hormones and stresses were analyzed using RT-qPCR. Result McUVR8 is 1 353 bp in length, encoding 450 amino acids, distributed in both nucleus and cytoplasm, with conserved helix-looped RCC1 core domain and C17 and C27 domains, and there is high homology with SsUVR8 of Salvia splendens, SdBUR8 of Salvia divinorum and ShUVR8 of Salvia hispanica, which are also in the Labiaceae family. McUVR8 was expressed in different tissues and leaf sequences of M. canadensis, with some differences, with the lowest expression in the roots, the highest expression in the flowers, and the highest expression in the first leaf in different leaf sequences. In addition, the McUVR8 gene responded to MeJA, ABA, and drought, NaCl, and two heavy metal and aluminum stress treatments in both leaves and roots. Finally, McUVR8 was screened and verified to interact with McCOP1. Conclusion McUVR8 may have important roles in regulating mint growth and development, responding to hormonal or adversity signals, and it may be involved in regulating mint secondary metabolism, adversity response, and other processes by binding to COP1, WRKYs, and other interacting proteins.

Key words: Mentha canadensis L, UV-B receptor McUVR8, protein characterization, protein interactions, expression pattern, raw letter analysis, gene cloning

KANG Qin, WANG Xia, SHEN Ming-yang, XU Jing-tian, CHEN Shi-lan, LIAO Ping-yang, XU Wen-zhi, WU Wei, XU Dong-bei. Cloning and Expression Analysis of the UV-B Receptor Gene McUVR8 in Mentha canadensis L.[J]. Biotechnology Bulletin, 2025, 41(8): 255-266.

Figures/Tables 10

Table 1 Information of primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	引物用途 Application
McUVR8-F	GAGAGAGATGGCCGACGAG	基因克隆
McUVR8-R	CTCAGATCCTCATTCTTTTTACGTC	基因克隆
McUVR8-GFP-F	CGGTACCCGGGGATCCATGGCCGACGAGGGGCA	蛋白定位
McUVR8-GFP-R	TGCTCACCATGTCGACGATCCTCATTCTTTTTACGTCAG	蛋白定位
qMcUVR8-F	GGCAGTGACGACTGATGGAA	表达分析
qMcUVR8-R	GCTGACCATTGGTACCCCTT	表达分析
β-actin-F	TGGAGAAAATATGGACAGAAAGTTG	表达分析
β-actin-R	CAGAAGAAGTTACTGAGTTGGGC	表达分析
BD-McUVR-F	GGAGGACCTGCATATGATGGCCGACGAGGGGCA	蛋白互作
BD-McUVR-R	GGATCCCCGGGAATTCTCAGATCCTCATTCTTTTTACGT

Fig. 1 McUVR8 cloning and protein structure analysisA: PCR amplification of McUVR8 (Red boxes and arrows denote the bands corresponding to the target genes. M: DNA-marker). B: Secondary structure prediction of McUVR8 (Blue: α-helix; purple: extended strand; orange: random coil). C: Tertiary structure prediction of McUVR8 (Red: extended strand; blue: random coil. Number 1‒7 indicate the lamellar structure consisting of seven WD40)

Fig. 2 Alignment of the amino acid sequences between McUVR8 and other plant UVR8 proteinsThe blue vertical lines indicate the seven WD40 structures; the green underline indicates the core structural domain of RCC1, and the red underlines indicate the C27 and C17 domains, respectively

Fig. 3 Motif analysis of UVR8 protein in different plantsConserved motif analysis of M. canadensis McUVR8 with homologous UVR8 proteins from 19 other species, the different species listed correspond to the 19 species in Fig. 2. The scale below this figure indicates amino acid sequence positions

Fig. 4 Phylogenetic tree analysis of UVR8 gene in different plantsPurple circles indicate M. canadensisMcUVR8, and green circles indicate the other 19 homologous UVR8 gene

Fig. 5 Subcellular localization analysis of McUVR8 protein of M. canadensis

Fig. 6 Tissue and phyllotaxis expression analysis of McUVR8 geneA: Expressions of McUVR8 in the root, stem, leaf, flower, bud, apical shoot of M. canadensis. B: Relative expressions of McUVR8 in the leaves at different positions. Leaf 1-leaf 8: The first to eighth leaf morphologically from top to bottom. Data are mean ± SE of 3 biological replicates, ANOVA is performed using Duncan's test, lowercase letters indicate significant differences at the 0.05 level, the same below

Fig. 7 McUVR8 gene in the leaves and roots in response to hormone and stress treatmentsExpression analysis of McUVR8 in the leaves of M. canadensis treated with MeJA (A), ABA (C), drought (E), and NaCl (G). Expression analysis of McUVR8 in the roots of M. canadensis treated with MeJA (B), ABA (D), drought (F), and NaCl (H)

Fig. 8 Expression profiles of McUVR8 gene in the leaves and roots in response to heavy metal or aluminum stressExpression analysis of McUVR8 in the leaves of M. canadensis treated with CdCl2 (A), CuCl2 (C), and AlCl3(E). Expression analysis of McUVR8 in the roots of M. canadensis treated with CdCl2 (B), CuCl2 (D), and AlCl3 (F)

Fig. 9 Y2H validations of protein interactions

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