月季类胡萝卜素裂解双加氧酶基因RcCCD4在花香合成中的功能

doi:10.16420/j.issn.0513-353x.2024-0382

摘要/Abstract

摘要：

为揭示类胡萝卜素裂解双加氧酶（carotenoid cleavage dioxygenase，CCDs）在花香合成通路中的作用，以中国古老月季‘月月粉’（Rosa chinensis‘Old Blush’）为材料，在花瓣中克隆了RcCCD4并进行功能解析。RT-qPCR结果表明，RcCCD4主要在花瓣中表达，花蕾期表达水平较低，盛开期表达水平最高。RcCCD4蛋白定位于细胞质体中。构建了RcCCD4的原核表达载体，通过酶活性检测发现，RcCCD4可以特异性地切割β-胡萝卜素。进一步分析发现，通过病毒诱导的基因沉默RcCCD4可显著降低花瓣中二氢-β-紫罗兰酮含量，而瞬时过表达RcCCD4可显著提高花瓣中二氢-β-紫罗兰酮含量。以上结果说明RcCCD4可以特异性地裂解β-胡萝卜素，进而合成二氢-β-紫罗兰酮。

关键词: 月季, 花香, 类胡萝卜素裂解双加氧酶, 原核表达, 病毒诱导的基因沉默

Abstract:

To reveal the role of carotenoid cleavage dioxygenases（CCDs）in biosynthetic pathway of floral scent，RcCCD4 gene were cloned from the petals of Rosa chinensis‘Old Blush’and functionally characterized. RT-qPCR showed that RcCCD4 was mainly expressed in petals，with the highest expression level at the fully opening stage and lowest at the bud stage. Subcellular localization assays showed that RcCCD4 were located in the plastid. Moreover，a prokaryotic expression protein vector for RcCCD4 were constructed and found that the RcCCD4 enzyme can specifically cleave β-carotene substrates in vitro enzymatic activity assay. Virus-induced gene silencing（VIGS）further suggested that silencing of RcCCD4 significantly reduced the content of dihydro-β-ionone in petals，and RcCCD4 overexpression led to increase in dihydro-β-ionone content. These results indicated that RcCCD4 can specifically cleave β-carotene substrates，thereby synthesizing dihydro-β-ionone.

Key words: rose, floral scent, carotenoid cleavage dioxygenase（CCD）, prokaryotic expression, virus induced gene silencing（VIGS）

付琪, 王丹, 景维坤, 张颢, 王慧纯, 蹇洪英, 邱显钦, 王其刚, 唐开学, 晏慧君. 月季类胡萝卜素裂解双加氧酶基因RcCCD4在花香合成中的功能[J]. 园艺学报, 2025, 52(3): 623-634.

FU Qi, WANG Dan, JING Weikun, ZHANG Hao, WANG Huichun, JIAN Hongying, QIU Xianqin, WANG Qigang, TANG Kaixue, YAN Huijun. Functional Characterization of Carotenoid Cleavage Dioxygenases 4 Gene（RcCCD4）Involved in Biosynthetic Pathway of Floral Scent in Rosa chinensis‘Old Blush’[J]. Acta Horticulturae Sinica, 2025, 52(3): 623-634.

图/表 9

表1 本研究中所用的引物

Table 1 Primers used in this study

用途 Function	引物名称 Primer name	引物序列（5′-3′）Primer sequence
克隆基因	RcCCD4-F	ATGGATGCCTTGTCTTCTTCT
Gene clone	RcCCD4-R	CAACTTCTTCAGGTCACTCTCC
病毒诱导的基因沉默	pTRV1-F	TTACAGGTTATTTGGGCTAG
Virus induced gene	pTRV1-R	CCGGGTTCAATTCCTTATC
silencing	pTRV2-F	TGGGAGATGATACGCTGTT
	pTRV2-R	CCTAAAACTTCAGACACG
	pTRV2-RcCCD4-F	GAGTAAGGTTACCGAATTCAGCTCGACGTGTCGG
	pTRV2-RcCCD4-R	CTCGAGACGCGTGAGCTCGGTACCATACAACAGTCACAACGTTTGTG
原核表达	Pet28a（+）-RcCCD4-F	CGCGGCAGCCATATGATGGATGCCTTGTCTTCTTCTT
Prokaryotic expression	Pet28a（+）-RcCCD4-R	GTGGTGGTGGTGCTCGAGCAACTTCTTCAGGTCACTCTCC
过表达	pSurper-1300-RcCCD4-F	AAGCTTCTGCAGGGGCCCGGGATGGATGCCTTGTCTTCTTCT
Overexpression	pSurper-1300-RcCCD4-R	TCCTCGCCCTTGCTCACCATGGTACCCAACTTCTTCAGGTCACTCTCC
实时荧光定量PCR qRT-PCR	qRT-RcCCD4-F	AGCTCGACGTGTCGGG
	qRT-RcCCD4-R	CCTCCGGATTCTCCGGATCC
	qRT-RcCCD1-F	GTTGATGTACGACAGTTCTCAGCCT
	qRT-RcCCD1-R	ACAAACTCTCCATTCAAGCAATCAGG
	qRT-RcCCD7-F	AGTGGAACAAGTCATCAGATTTTCCAGT
	qRT-RcCCD7-R	TAGTTGAAGCATTCAGCTTCACCAC
	qRT-RcCCD8-F	TGTTTTAATGACATGCCGCCTTGAG
	qRT-RcCCD8-R	ATAATTTCTTCTGTGTTGCTAAACCGGT
	qRT-RcZAS-F	GAGGGTGAAGCGATTGCAAAAATT
	qRT-RcZAS-R	TGACACGGAGAAAATTCATGGTTACAG
	qRT-RcZAS-like-F	TGCCAACCTCATCGAACACCTAA
	qRT-RcZAS-like-R	ATGGTCAACTACACACTTGTATACGACT
	qRT-RcNCED1-F	CAATCTCACTGAACCCACTTGCTT
	qRT-RcNCED1-R	CACTACTACTTTAACTCAAAGCCTGCTCC
	qRT-RcNCED6-F	AGAAGACGTGGAAGTCGGAGC
	qRT-RcNCED6-R	AGCTACTGCTATGCCTGGTTTTTCA

图1 月季RcCCD4与其他物种系统发育进化树

Fig. 1 Phylogenetic trees of rose RcCCD4 and other species

图2 月季RcCCD4在烟草叶片细胞中的亚细胞定位

Fig. 2 Subcellular localization of rose RcCCD4 in leaf cells of Nicotiana benthamiana

图3 ‘月月粉’月季CCD家族基因在不同组织中的表达以叶片为对照进行比较，t-test，*代表显著性水平α = 0.05；**代表显著性水平α = 0.01；ns：无显著性

Fig. 3 Expression of CCD family genes in different organs of Rosa chinensis‘Old Blush’ Compared with leaf as a control，t-test，* represents a significance level of α = 0.05；** represents a significance level of α = 0.01；ns：No significant

图4 ‘月月粉’月季RcCCD4在不同开放时期花瓣中的表达以花蕾期S1级为对照进行比较，t-test，*代表显著性水平α = 0.05；**代表显著性水平α = 0.01；ns：无显著性

Fig. 4 Expression of RcCCD4 in the petals during different developmental stages of Rosa chinensis‘Old Blush’ Compared with S1 stage in the bud stage as a control，t-test，* represents a significance level of α = 0.05；** represents a significance level of α = 0.01；ns：No significant

图5 RcCCD4融合蛋白诱导条件优化（A）和Western-Blot印迹（B） M：Marker。箭头指示目的蛋白；1 ~ 4：不同温度及不同浓度IPTG诱导样品；5：未诱导样品；6、8、10、12：沉淀样品；7、9、11、13：上清液样品

Fig. 5 Conditions optimization（A）and Western-Blot analysis（B）of RcCCD4 recombination protein M：Marker. The arrows indicate the protein of interest；1-4：IPTG-induced samples at different temperatures and concentrations；5：Uninduced sample；6，8，10，12：Precipitated sample；7，9，11，13：Supernatant samples

图6 RcCCD4在大肠杆菌中的表达分析（A）和β-类胡萝卜素HPLC检测结果（B）

Fig. 6 Expression analysis of RcCCD4 in Escherichia coli（A）and HPLC detection of β-carotenoids（B）

图7 ‘洛神’月季RcCCD4沉默株系花瓣中基因相对表达量和二氢-β-紫罗兰酮含量 t-test，*代表显著性水平α = 0.05；**代表显著性水平α = 0.01；ns：无显著性；NT：未处理样本，Mock：TRV空载样本。下同

Fig. 7 Expression of RcCCD4 in infected plants by qRT-PCR and relative contents of dihydro-β-ionone in Rosa hybrida‘Luoshen’ t-test，* represents a significance level of α = 0.05；** represents a significance level of α = 0.01；ns：No significant；NT：No-treatment sample；Mock：TRV empty sample. The same below

图8 ‘月月粉’和‘洛神’月季RcCCD4过表达株系花瓣中基因表达量和二氢-β-紫罗兰酮含量

Fig. 8 Expression of RcCCD4 in overexpression plants by qRT-PCR and relative contents of dihydro-β-ionone in Rosa chinensis‘Old Blush’and Rosa hybrida‘Luoshen’

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