薄荷UV-B受体基因McUVR8的克隆与表达分析

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

摘要/Abstract

摘要：

目的紫外（UV-B）受体UVR8在调控植物生长发育、响应逆境胁迫等过程中发挥关键作用。探究薄荷（Mentha canadensis L.）UV-B受体基因McUVR8的蛋白特性和表达特征，预测其互作蛋白，为后续探究McUVR8功能提供理论参考。方法通过RT-PCR技术获得McUVR8基因，利用多种生物信息学工具对其理化性质、蛋白结构和亚细胞定位、互作蛋白进行分析预测。利用RT-qPCR分析McUVR8的组织、叶序表达模式及其在不同激素、胁迫下的表达变化。结果 McUVR8全长1 353 bp，编码450个氨基酸，在细胞核和细胞质中均有分布，具有保守的呈螺旋环状的RCC1核心结构域和C17、C27结构域，与同为唇形科的一串红（Salvia splendens）SsUVR8、迷幻鼠尾草（Salvia divinorum）SdBUR8和西班牙鼠尾草（Salvia hispanica）ShUVR8同源性较高。McUVR8在薄荷不同组织和叶序中均表达，并具有一定差异，其中在根中表达量最低，花中表达量最高，不同叶序中其表达在第一片叶中最高。此外，McUVR8基因在叶片和根中均响应MeJA、ABA以及干旱、NaCl和2种重金属和铝胁迫处理。最后，筛选并验证到McUVR8与McCOP1互作。结论 McUVR8可能在调节薄荷的生长发育、响应激素或逆境信号中具有重要作用，并且其可能通过与COP1、WRKY等蛋白互作，参与调控薄荷次生代谢、逆境响应等过程。

关键词: 薄荷, UV-B受体McUVR8, 蛋白特征, 蛋白互作, 表达模式, 生信分析, 基因克隆

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

康琴, 汪霞, 谌明洋, 徐静天, 陈诗兰, 廖平杨, 许文志, 吴卫, 徐东北. 薄荷UV-B受体基因McUVR8的克隆与表达分析[J]. 生物技术通报, 2025, 41(8): 255-266.

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.

图/表 10

表1 本研究所使用的引物信息

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

图1 McUVR8克隆及蛋白结构分析A：McUVR8的PCR扩增（红色方框和箭头指示目的基因条带，M：DNA-marker）；B：McUVR8的二级结构预测（蓝色：α螺旋；紫色：延伸链；橙色：随机卷曲）；C：McUVR8的三级结构预测（红色：延伸链；蓝色：随机卷曲；数字1‒7表示7个WD40组成的片层结构）

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)

图2 McUVR8氨基酸序列与其他植物UVR8蛋白的氨基酸序列比对蓝色垂直线表示7个WD40结构；绿色下划线条所示部分代表RCC1核心结构域，红色下划线条所示部分分别代表C27结构域和C17结构域

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

图3 不同植物UVR8蛋白的motif分析薄荷McUVR8与其他19个物种中同源UVR8蛋白的保守motif分析，所列不同物种与图2中的19个物种一致。图片下方标尺表示氨基酸序列位置

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

图4 不同植物的UVR8基因的系统进化树分析紫色圆圈表示薄荷McUVR8，绿色圆圈表示其他19个同源UVR8基因

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

图5 薄荷McUVR8蛋白的亚细胞定位分析Bar=50 μm

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

图6 McUVR8基因的组织和叶序表达分析A：McUVR8在薄荷根、茎、叶、花、蕾、茎尖中的表达；B：McUVR8在薄荷不同叶片中的表达，叶1-叶8：形态学从上到下的第1至第8片叶子；数据为3个生物学重复的平均值（mean）± SE，方差分析采用邓肯检验，小写字母代表0.05水平上差异显著，下同

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

图7 McUVR8基因在叶片和根中响应激素和胁迫处理McUVR8在MeJA（A）、ABA（C）、干旱（E）、NaCl（G）处理的薄荷叶片中的表达情况；McUVR8在MeJA（B）、ABA（D）、干旱（F）、NaCl（H）处理的薄荷根中的表达情况

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)

图 8 McUVR8基因在叶片和根中响应重金属或铝胁迫的表达模式McUVR8在CdCl2（A）、CuCl2（C）、AlCl3（E）处理的薄荷叶片中的表达情况；McUVR8在CdCl2（B）、CuCl2（D）、AlCl3（F）处理的薄荷根中的表达情况

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)

图9 Y2H验证蛋白互作

Fig. 9 Y2H validations of protein interactions

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