紫丁香SoCHR35参与罗勒烯合成的功能解析

doi:10.16420/j.issn.0513-353x.2024-0817

摘要/Abstract

摘要：

为探究RNA介导的DNA甲基化途径中染色质重塑复合体ATP酶35（Chromatin remodeling complex ATPase 35，CHR35）在紫丁香（Syringa oblata）花香成分罗勒烯合成中的作用，克隆了SoCHR35，并进行了生物信息学、时空表达模式分析、功能验证和亚细胞定位。结果显示，SoCHR35的CDS序列全长2 073 bp，编码690个氨基酸；该蛋白含有典型的SNF2保守结构域DEXDc和HELICc，与芳香植物紫苏（Perilla frutescens）和芝麻（Sesamum indicum）等聚类在同一分支。时空表达分析表明，该基因表达随花发育表现出先降低后升高趋势，与花香释放呈负相关。在紫丁香花瓣中瞬时过表达该基因，罗勒烯释放量显著降低；瞬时沉默该基因，罗勒烯释放量则显著升高。通过亚细胞定位发现，SoCHR35定位于叶绿体，与罗勒烯合成酶SoTPS2表达定位相同；瞬时沉默SoCHR35后，花瓣中SoTPS2的表达水平显著升高。结果表明，SoCHR35负调控紫丁香罗勒烯的合成，影响花香释放，且可能是通过直接或间接调控SoTPS2发挥作用。

关键词: 紫丁香, 罗勒烯, SoCHR35, DNA甲基化, 花香

Abstract:

In order to investigate the role of SoCHR35（Chromatin remodeling complex ATPase 35），a member of RNA-mediated DNA methylation pathway，in the biosynthesis of lilac floral substance—ocimene，the SoCHR35 gene was cloned and analyzed，including bioinformatics，spatiotemporal expression pattern，functional characterization，and subcellular localization. The results showed that the total length of the CDS sequence of SoCHR35 was 2 073 bp，encoding for 690 amino acids. The protein had the typical SNF2 conserved domains of DEXDc and HELICc，and was clustered with CHR35 of Perilla frutescens and Sesamum indicum. The spatiotemporal expression analysis showed that with the flower development the expression level of SoCHR35 first decreased and then increased，which was negatively correlated with the release of floral scents. After transiently overexpressing SoCHR35 in lilac petals，the release amount of ocimene was significantly reduced，while transiently silencing of SoCHR35 lead to a remarkable increase in the release amount of ocimene. Through subcellular localization，SoCHR35 was found to be located in chloroplasts，which was same to the expression position of SoTPS2，a ocimene synthase gene. Moreover，the transient silencing of SoCHR35 resulted in a significant enhancement of SoTPS2 expression in petals. These results indicated that SoCHR35 negatively regulated the biosynthesis of ocimene in lilac petals to impact the floral scent release，which may result from a direct or indirect regulation of SoTPS2.

Key words: Syringa oblata, ocimene, SoCHR35, DNA methylation, floral scent

汪进萱, 倪莹, 孟昕, 冷卓, 马波, 冷平生, 吴静, 胡增辉. 紫丁香SoCHR35参与罗勒烯合成的功能解析[J]. 园艺学报, 2025, 52(10): 2655-2665.

WANG Jinxuan, NI Ying, MENG Xin, LENG Zhuo, MA Bo, LENG Pingsheng, WU Jing, HU Zenghui. Functional Analysis of SoCHR35 in the Biosynthesis of Ocimene in Syringa oblata[J]. Acta Horticulturae Sinica, 2025, 52(10): 2655-2665.

图/表 7

表1 本研究中所用到的引物序列

Table 1 Primer sequences used in this study

引物用途 Primer function	引物名称 Primer name	引物序列（5′-3′） Primer sequence
克隆基因 Gene clone	SoCHR35-F	ATGGTTAATGACATTGTTCTACATGT
克隆基因 Gene clone	SoCHR35-R	CCTTTTGAAGACAGCAACCACAT
实时荧光定量PCR qRT-PCR	SoActin-F	TGGAATGTGCTGAGAGATGC
	SoActin-R	TGCTGACCGTATGAGCAAAG
	qSoCHR35-F	AATTTTCCCACATCCACGGC
	qSoCHR35-R	TTCCAGAACCAGGTCATGA
	qSoTPS2-F	GGTTTGCTTCAGGGCACTTG
	qSoTPS2-R	CATCCACCTGAACGGTTCCA
基因过表达 Gene overexpression	PRI101-SoCHR35-F	ATGCCCGTCGACCCCGGGATGGTTAATGACATTGTTCTACATGTTGGC
基因过表达 Gene overexpression	PRI101-SoCHR35-R	TGCTCACCATGGATCCTTACCTTTTGAAGACAGCAACCACATCT TGGGAGATGATACGCTGTT
基因沉默 Gene silencing	TRV2-SoCHR35-F	GTCCAGTCCTGGCCTCGTCGGCCGATAACTATAAGAGGAAGGTTACCGTAATACAAGATC
	TRV2-SoCHR35-R	GACCACAAGTGGCCAGACTGGCCTAAGAAATTCTTCAACTTTGGGTGAACATAAACTGC
	TRV1-F	TTACAGGTTATTTGGGCTAG
	TRV1-R	CCGGGTTCAATTCCTTATC

图1 SoCHR35与其他物种CHR35蛋白序列比对分析黑色、红色、蓝色部分分别表示同源性 = 100%、≥ 75%、≥ 50%；红色框为DEXDc保守基序，绿色框为HELICc保守基序。Oe：油橄榄；Si：芝麻；Ac：中华猕猴桃；Ap：穿心莲；Ca：辣椒；Ce：咖啡

Fig. 1 Alignment and analysis of SoCHR35 with CHR35 protein sequences of other species The black，red，and blue parts represent homology = 100%，≥ 75%，≥ 50%，respectively. Red boxes are DEXDc conserved motifs，and green boxes are HELICc conserved motifs. Oe：Olea europaea；Si：Sesamum indicum；Ac：Actinidia chinensis；Ap：Andrographis paniculata；Ca：Capsicum annuum；Ce：Coffea eugenioides

图2 紫丁香SoCHR35与其他植物同源蛋白的系统发育树

Fig. 2 Phylogenetic tree of SoCHR35 from Syringa oblata and homologous proteins of other plant species

图3 SoCHR35在不同器官（A）和不同花发育阶段（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. 3 The expression pattern of SoCHR35 in different organs（A）and at different floral developmental stages（B） Different lowercase letters indicate significant differences at the P < 0.05 level

图4 瞬时过表达SoCHR35对花香的影响 A：基因过表达效率；B：花香释放量；C：35S和35S:SoCHR35花瓣的香气化合物总离子色谱图

Fig. 4 The effect on floral scent of SoCHR35 transient overexpression A：Gene overexpression efficiency；B：The release amount of floral scents；C：Total ion current（TIC）chromatograms of the floral scent compounds emitted from lilac of 35S and 35S:SoCHR35 t-test，**P < 0.01，***P < 0.001

图5 VIGS瞬时沉默SoCHR35对花香的影响 A：基因沉默效率；B：花香释放量；C：TRV和TRV:SoCHR35花瓣的香气化合物总离子色谱图

Fig. 5 The effect on floral scent of SoCHR35 transient silencing A：Gene silencing efficiency；B：The release amount of floral；C：Total ion current（TIC）chromatograms of the floral scent compounds emitted from lilac of TRV and TRV:SoCHR35 t-test，** P < 0.01，**** P < 0.0001

图6 SoCHR35蛋白的亚细胞定位（A）及其对下游SoTPS2的表达分析（B）

Fig. 6 The subcellular localization of SoCHR35（A）and the expression analysis of downstream SoTPS2（B） t-test，*** P < 0.001

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