牡丹PsMYB43调控芳樟醇合成的功能解析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0832

摘要/Abstract

摘要：

目的单萜物质芳樟醇是牡丹‘黄冠’花香的重要组分，MYB类转录因子在单萜物质合成中发挥着重要作用。探究PsMYB43在牡丹芳樟醇合成中的作用，为牡丹花香的遗传改良提供基因储备与理论依据。方法以牡丹‘黄冠’为试材，首先克隆PsMYB43基因的CDS序列，利用生物信息学软件对其理化性质、保守结构域及系统进化关系进行分析；其次，通过RT-qPCR分析其在不同花发育时期和器官组织中的表达模式；利用烟草进行亚细胞定位，最后通过瞬时过表达和瞬时沉默PsMYB43进行功能验证，以明确PsMYB43在单萜物质芳樟醇合成中的作用。结果 PsMYB43的CDS序列长1 140 bp，编码379个氨基酸，PsMYB43为不稳定的亲水蛋白，具有2个不完全重复的HTH结构，属于R2R3型MYB转录因子。系统进化分析显示，牡丹PsMYB43蛋白与滇牡丹PdMYB12L蛋白的亲缘关系最近。随着花发育，PsMYB43的表达水平先升高后降低，在半开期达到顶峰，与芳樟醇释放规律一致；PsMYB43在盛开期外瓣中表达量最高，而在心皮和雄蕊中表达水平相对较低。亚细胞定位表明PsMYB43定位于细胞核。金鱼草花瓣中瞬时过表达PsMYB43后，单萜物质罗勒烯、月桂烯和芳樟醇含量明显升高，分别升高了2.0、1.67和3.4倍；牡丹花瓣中瞬时沉默PsMYB43后，芳樟醇释放量显著降低，下降了38%。结论 PsMYB43对牡丹花瓣中单萜物质芳樟醇的合成具有正调控作用。

关键词: 牡丹, 花香, MYB转录因子, 芳樟醇, 功能验证

Abstract:

Objective The monoterpene substance linalool is an important component of the floral fragrance in tree peony (Paeonia suffruticosa ‘OuKan’), and MYB-type transcription factors play a crucial role in the synthesis of monoterpenes. Exploring the role of PsMYB43 in the synthesis of linalool in tree peony, aiming to provide genetic resources and theoretical basis for genetic improvement of floral fragrance. Method Using the P. suffruticosa ‘OuKan’ as the experimental material, the CDS sequence of the PsMYB43 gene was first cloned, and its physicochemical properties, conserved structural domains, and phylogenetic relationships were analyzed using bioinformatics software. Secondly, RT-qPCR was used to analyze its expression patterns in different floral development stages and organ tissues. Subcellular localization was performed using tobacco, and finally the function of PsMYB43 was validated through transient overexpression and transient silencing to clarify its role in the synthesis of monoterpene compound linalool. Result The CDS sequence of PsMYB43 is 1 140 bp and encodes 379 amino acids. PsMYB43 is an unstable hydrophilic protein with two incomplete HTH domains, belonging to the R2R3-type MYB transcription factor family. Systematic evolutionary analysis revealed that the PsMYB43 protein in P. suffruticosa is most closely related to the PdMYB12L protein in P. delavayi. With flowering developing, the expressions first increased and then decreased, reaching its peak at the half-opening stage, consistent with the release pattern of linalool. PsMYB43 has the highest expressions in the outer petals during full blooming, while its expressions relatively low in carpels and stamens. Subcellular localization indicated that PsMYB43 is localized in the cell nuclear. Following transient overexpression of PsMYB43 in snapdragons, the levels of monoterpenes such as linalool, myrcene, and linalool significantly increases, rising by 2.0, 1.67, and 3.4-fold, respectively. Following transient silencing of PsMYB43 in tree peony, linalool release decreases significantly by 38%. Conclusion PsMYB43 positively regulates the synthesis of monoterpenoid linalool in tree peony petals.

Key words: Paeoniasuffruticosa, floral fragrance, MYB transcription factor, linalool, functional validation

王振泉, 钟毅, 马波, 吴静, 胡增辉. 牡丹PsMYB43调控芳樟醇合成的功能解析[J]. 生物技术通报, 2026, 42(4): 263-271.

WANG Zhen-quan, ZHONG Yi, MA Bo, WU Jing, HU Zeng-hui. Functional Analysis of PsMYB43 Regulating Linalool Synthesis in Tree Peony[J]. Biotechnology Bulletin, 2026, 42(4): 263-271.

图/表 8

图1 ‘黄冠’牡丹7个不同花发育时期及盛开期的不同器官组织

Fig. 1 Seven different flower developmental stages and different organ tissues at full blooming in P. suffruticosa 'QuKan'

表1 本研究中所用引物序列

Table 1 Sequences of primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	作用 Function
PsMYB43-F	ATGGACTCTGTTCCGTCCAAG	基因克隆 Gene clone
PsMYB43-R	TCAAGAAAGAAGCCAAGCAAC	基因克隆 Gene clone
qPsMYB43-F	TTGACGGTGACTGGTAATG	实时荧光定量 RT-qPCR
qPsMYB43-R	CCAATCAATCCTTCCATCATC	实时荧光定量 RT-qPCR
yPsMYB43-F	ACCTCGACTCTAGAAATGGACTCTGTTCCGTCCAAGA	亚细胞定位 Subcellular localization
yPsMYB43-R	TTTTTCTACCGGTACAGAAAGAAGCCAAGCAACCATAG	亚细胞定位 Subcellular localization
gPsMYB43-F	ATGCCCGTCGACCCCATGGACTCTGTTCCGTCCAAGA	瞬时过表达 Transient overexpression
gPsMYB43-R	CCATGGATCCGGTACAGAAAGAAGCCAAGCAACCATAG	瞬时过表达 Transient overexpression
vPsMYB43-F	TGAGTAAGGTTACCGGAAGAGCTGGAAAATGGGATGTTAT	瞬时沉默 Transient silencing
vPsMYB43-R	GTGAGCTCGGTACCGCTTCCAAATCCCAATCAATCCTTC	瞬时沉默 Transient silencing
PsACTIN-F	GCGTTGACGACTTTCTCATCCT	牡丹荧光定量内参 Internal reference gene of tree peony
PsACTIN-R	CGCACATCCTTGACAAACCCTA	牡丹荧光定量内参 Internal reference gene of tree peony
AmUBI-F	GACCTATACCAAGCCGAAG	金鱼草荧光定量内参 Internal reference gene of snapdragon
AmUBI-R	CGTTCCAGCACCACAATC	金鱼草荧光定量内参 Internal reference gene of snapdragon

图2 PsMYB43与同源蛋白序列比对分析深蓝：同源性100%；红色：同源性≥75%；蓝色：同源性≥50%

Fig. 2 Sequence alignment analysis of PsMYB43 with homologous proteinsDark blue: Homology 100%. Red: Homology≥75%. Blue: Homology≥50%

图3 PsMYB43的系统发育树分支上数字代表bootstrap值

Fig. 3 Phylogenetic analysis of PsMYB43The number on the branch are the bootstrap value (%)

图4 PsMYB43表达模式A：PsMYB43在7个不同花发育时期的相对表达水平；B：PsMYB43在盛开期8个不同器官组织中的表达水平。不同小写字母表示在P<0.05水平差异显著

Fig. 4 Expression patterns of PsMYB43 geneA: Relative expressions of PsMYB43 during seven flower development stages. B: Relative expressions of PsMYB43 in eight different organs and tissues during full blooming stage. Different lowercase letters indicate significant differences at P<0.05 level

图5 牡丹PsMYB43的亚细胞定位

Fig. 5 Subcellular localization of PsMYB43 in P. suffruticosa

图6 过表达PsMYB43基因表达量检测、对不同单萜物质的影响及GC-MS分析A：PsMYB43基因表达量；B：单萜物质释放量；C：GC-MS分析挥发性物质；ns P>0.5；** P<0.01；*** P<0.001；**** P<0.000 1，下同

Fig. 6 Detection of the expression of overexpressed PsMYB43, its effect on different monoterpene substances, and GC-MS analysisA: PsMYB43 gene expression. B: Release of monoterpenoid substances. C: GC-MS analysis of VOCS. ns P>0.5, ** P<0.01, *** P<0.001, and **** P<0.000 1, the same below

图7 沉默PsMYB43表达量检测（A）及对芳樟醇的影响（B）

Fig. 7 Detection of silencing PsMYB43 expression (A) and its effect on linalool (B)

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