‘西伯利亚’百合LiMYB4的克隆及在萜烯合成中的功能分析

doi:10.16420/j.issn.0513-353x.2025-0946

摘要/Abstract

摘要：

萜烯类物质是百合（Lilium spp.）花香的主要成分，MYB类转录因子在调控萜烯合成中发挥着重要作用。以‘西伯利亚’百合（Lilium‘Siberia’）为材料，克隆LiMYB4基因，并进行时空表达特异性、同源蛋白比对与系统发育分析，最后通过瞬时沉默与过表达验证其在百合萜烯合成中的功能。结果显示，LiMYB4的开放阅读框（open reading frame，ORF）序列为738 bp，编码245个氨基酸；LiMYB4具有2个典型的MYB保守结构域，为R2R3-MYB亚家族成员，与老鸦瓣（Amana edulis）AeMYB4蛋白的相似性最高（71.26%）；LiMYB4在内、外花被片中的表达水平显著高于根、茎和叶，随花发育开放呈先降低后升高趋势，并与萜烯释放规律相反；沉默LiMYB4后，‘西伯利亚’百合花瓣中主要萜烯类物质芳樟醇与罗勒烯的释放量均显著升高，而在过表达LiMYB4的花瓣中显著降低；分析萜烯骨架合成基因表达发现，沉默LiMYB4花瓣中合成基因LiDXR、LiGPPS、LiOcS、LiLiS的表达水平显著升高，过表达LiMYB4后则明显降低。以上结果表明，LiMYB4通过负调控萜烯合成通路基因的表达，抑制了‘西伯利亚’百合萜烯类物质的合成。

关键词: 百合, 花香, 萜类物质, LiMYB4, 功能

Abstract:

Terpenoids constitute the primary volatile components of lily floral scent，and MYB transcription factors are crucial regulators of terpenoid biosynthesis. In this study，Lilium‘Siberia’was used as the experimental material for cloning and characterizing LiMYB4，including analysis of spatiotemporal expression patterns，protein sequence homology，and phylogenetic relationships. In addition，transient silencing and overexpression assays were employed in the petals to investigate the role of LiMYB4 in terpenoid biosynthesis. The results showed that LiMYB4 contains a 738 bp ORF encoding 245 amino acids. The predicted LiMYB4 possesses two conserved MYB domain repeats and was thus classified as a member of the R2R3-MYB subfamily. The phylogenetic analysis showed the highest similarity（71.26%）with AeMYB4 from Amana edulis. The spatial expression profiling demonstrated significantly higher transcript level of LiMYB4 in perianths compared with roots，stems，or leaves. During floral development LiMYB4 expression first decreased and then increased，which was inversely correlated with terpenoid emission dynamics. The functional validation showed that transient silencing of LiMYB4 increased the emission of linalool and ocimene in petals. Conversely，LiMYB4 overexpression markedly reduced the emission of these volatiles. Further analysis of the terpene backbone biosynthesis genes revealed that the transcript levels of LiDXR，LiGPPS，LiOcS，and LiLiS were significantly upregulated in LiMYB4-silenced petals，but downregulated in LiMYB4-overexpressing petals. Collectively，these results demonstrate that LiMYB4 acts as a negative regulator of terpenoid biosynthesis in Lilium‘Siberia’by directly or indirectly suppressing the expression of key genes in the terpenoid biosynthesis pathway.

Key words: Lilium, floral scent, terpenoids, LiMYB4, function

沈言, 夏子轶, 冷平生, 马波, 胡增辉. ‘西伯利亚’百合LiMYB4的克隆及在萜烯合成中的功能分析[J]. 园艺学报, 2026, 53(2): 525-537.

SHEN Yan, XIA Ziyi, LENG Pingsheng, MA Bo, HU Zenghui. Cloning and Functional Analysis of LiMYB4 in Regulating Terpenoid Biosynthesis in Lilium‘Siberia’[J]. Acta Horticulturae Sinica, 2026, 53(2): 525-537.

图/表 10

图1 ‘西伯利亚’百合不同花发育时期（A）与盛开期器官组织（B）

Fig. 1 Different floral development stages（A），organs and tissues（B）in full flowering stage of Lilium‘Siberia’

表1 本研究中所用引物

Table 1 Primers used in this study

引物用途 Primer function	引物名称 Primer name	序列（5′-3′） Primer sequence
基因克隆 Gene clone	LiMYB4-QCF	ATGGTGAGAGCTCCTTGCTGTGA
基因克隆 Gene clone	LiMYB4-QCR	TCAGATTGTGGGCAAGTCCTGCA
亚细胞定位 Subcellular localization	LiMYB4-F	CAGTGGTCTCACAACATGGTGAGAGCTCCTTGCTG
亚细胞定位 Subcellular localization	LiMYB4-R	CAGTGGTCTCATACAGATTGTGGGCAAGTCCTGCA
实时荧光定量PCR qRT-PCR	qLiMYB4-F	AAATCTGAAGCACCCCACATT
实时荧光定量PCR qRT-PCR	qLiMYB4-R	CATCCACCATGCGAGTATGAT
基因沉默 Gene silencing	pTRV2-LiMYB4-EcoR Ⅰ-F	TTAGATTCTGTGAGTAAGGTTACCGATGGTGAGAGCTCCTTGCTG
基因沉默 Gene silencing	pTRV2-LiMYB4-Xho Ⅰ-R	AATGTCTTCGGGACATGCCCGGGCCACTATAGATGATTCGGTGGCCG
基因过表达 Gene overexpression	35S-LiMYB4-Sfi I-F	GTCCAGTCCTGGCCTCGTCGGCCATGGTGAGAGCTCCTTGCTG
基因过表达 Gene overexpression	35S-LiMYB4-Sfi I-R	GACCACAAGTGGCCAGACTGGCCGATTGTGGGCAAGTCCTGCAG

图2 LiMYB4基因克隆 A：‘西伯利亚’百合总RNA提取电泳检测图，M1：Trans2K Plus DNA marker；B：LiMYB4基因克隆电泳图，M2：DL2000 DNA Marker

Fig. 2 Cloning of LiMYB4 gene A：Agarose gel electrophoresis detection of total RNA in Lilium‘Siberia’；M1：Trans2K Plus DNA marker；B：Agarose gel electrophoresis of LiMYB4 gene；M2：DL2000 DNA marker

图3 LiMYB4与其他植物MYB4蛋白同源性比对分析 Lr：岷江百合；Ae：老鸦瓣；Cn：椰子；Tl：宽叶香蒲。蓝色、黄色、绿色分别表示同源性为100%、≥ 75%、≥ 50%；黑色横线标记为两个MYB保守结构域

Fig. 3 Homology alignment analysis of LiMYB4 with MYB4 from other plants Lr：Lilium regale；Ae：Amana edulis；Cn：Cocos nucifera；Tl：Typha latifolia. The blue，yellow，and green parts represent homology = 100%，≥ 75%，≥ 50%，respectively. Black horizontal lines mark two MYB conserved domains

图4 LiMYB4与其他植物MYB4的系统进化树

Fig. 4 Phylogenetic tree of LiMYB4 and MYB4 from other species

图5 LiMYB4在‘西伯利亚’百合不同花发育阶段（A）和不同器官组织（B）的表达模式不同小写字母表示在P < 0.05水平上显著差异

Fig. 5 The expression patterns of LiMYB4 at different floral developmental stages（A）and in different organs and tissues（B）of Lilium‘Siberia’ Different lowercase letters indicate significant differences at the P < 0.05 level

图6 LiMYB4的亚细胞定位 A：拟南芥原生质体，红色荧光为叶绿体自发荧光；B：烟草叶片，红色荧光为细胞核Marker

Fig. 6 Subcellular localization of LiMYB4 A：Arabidopsis protoplast，with red fluorescence representing chloroplast autofluorescence；B：Nicotiana benthamiana leaf，with red fluorescence indicating nuclear marker

图7 瞬时沉默LiMYB4对‘西伯利亚’百合萜烯释放量的影响 A：基因沉默效率；B：两种主要萜烯化合物的释放量；C：TRV和TRV-LiMYB4花香化合物代表性总离子色谱图。

Fig. 7 Effect of silencing LiMYB4 on terpenoid emission in Lilium‘Siberia’ A：Gene silencing efficiency；B：The release amount of the two main monoterpenoids；C：Representative total ion chromatograms of the floral scent compounds from TRV and TRV-LiMYB4. t-test，* P < 0.05，****P < 0.0001

图8 瞬时过表达LiMYB4对‘西伯利亚’百合萜烯释放量的影响 A：基因过表达效率；B：两种主要萜烯化合物的释放量；C：35S和35S-LiMYB4花香化合物代表性总离子色谱图。

Fig. 8 Effect of overexpression LiMYB4 on terpenoid emission in Lilium‘Siberia’ A：Gene overexpression efficiency；B：The release amount of the two main monoterpenoids；C：Representative total ion chromatograms of the floral scent compounds from 35S and 35S-LiMYB4. t-test，**P < 0.01，****P < 0.0001

图9 瞬时沉默（A）和过表达（B）LiMYB4对萜烯合成通路基因LiDXR、LiGPPS、LiOcS和LiLiS表达的影响

Fig. 9 Effects of transient silencing（A）and overexpression（B）of LiMYB4 on the expression of terpenoid biosynthesis pathway genes LiDXR，LiGPPS，LiOcS，and LiLiS t-test，*P < 0.05，**P < 0.01，****P < 0.0001

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