澳洲石斛萜类合成酶基因DkTPS7的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0403

摘要/Abstract

摘要：

【目的】萜类合成酶（terpene synthase, TPS）是参与萜类物质合成的关键酶，在植物花香形成中具有重要作用。探究TPS基因在石斛花香气形成中的作用，为进一步了解不同时期石斛花香气的动态变化及其形成机制提供参考。【方法】采用顶空固相微萃取方法结合气相色谱-质谱联用技术，比较不同时期澳洲石斛花的香气成分差异；利用RT-PCR技术克隆DkTPS7，并进行生物信息学分析；利用农杆菌介导的瞬时转化系统检测其亚细胞定位；利用RT-qPCR技术检测其在不同品种、不同花发育时期和日变化中的表达模式。【结果】澳洲石斛中共检测到523种香气物质，萜类化合物是该品种香气中最丰富的挥发物。DkTPS7的开放阅读框（open reading frame, ORF）为1 797 bp，编码598个氨基酸，含有3个萜类合成酶家族的保守结构域，属于TPS-b亚家族；亚细胞定位分析显示，DkTPS7定位于质体。RT-qPCR结果显示，DkTPS7的表达具有品种特异性，在澳洲石斛中高表达，‘杂交紫花’和麝香品种中几乎不表达；DkTPS7在始花期的澳洲石斛花中的表达量最高，盛花期次之；1 d中，DkTPS7基因表达量变化呈现先升后降的趋势。【结论】从澳洲石斛花中克隆得到DkTPS7基因，其表达具有品种和时空特异性，且其表达模式与单萜类香气物质的积累趋势一致。

关键词: 石斛, 顶空固相微萃取-气相色谱-质谱联用法（HS-SPME-GC-MS）, 香气, 萜类合成酶, 基因克隆, 亚细胞定位, 表达分析

Abstract:

【Objective】 Terpene synthase（TPS）is a key enzyme in the biosynthesis of terpenoids, which possesses an important role in the formation of plant flower fragrance. Exploring the role of TPS gene in formation of Dendrobium flower fragrance may provide references for the dynamic changes and related formation mechanism of Dendrobium flower fragrance at different flowering stages. 【Method】 Headspace solid phase microextraction-gas chromatograohy-mass spectrometry（HS-SPME-GC-MS）was used to compare the difference in the fragrance components during flowering stages of Dendrobium kingianum. RT-PCR was applied to clone a DkTPS7 gene, and its bioinformatics analysis was conducted. The agrobacterium-mediated transformation was to detect their subcellular localizations. RT-qPCR technology was to detect the expression profiles in different varieties, during flowering stages, and on diurnal variations. 【Result】 A total of 523 frangrance components were identified. Terpenoids was the most abundant volatiles constituting the fragrance of this variety. DkTPS7 included a 1 797 bp open reading frame for 598 amino acids. The DkTPS7 contained three conserved domains and belonged to TPS-b subfamily. Subcellular localization analysis revealed that DkTPS7 was localized in the plastid. The result of RT-qPCR analysis showed that DkTPS7 was highly expressed in Dendrobium kingianum, while it has almost no expression in Dendrobium ‘Zajiaozihua’ and Dendrobium parishii. The expression of DkTPS7 was the highest at the half flowering stage of Dendrobium kingianum, followed by the bud stage and the lowest at the bud stage. The expression of DkTPS7 also showed a trend of raising first and then decreasing. 【Conclusion】 DkTPS7 is cloned from D. kingianum. The expression of DkTPS7 has variety and temporospatial characters, and the expression pattern is consistent with the accumulation trend of monoterpenoid.

Key words: Dendrobium headspace solid phase microextraction-gas chromatograohy-mass spectrometry（HS-SPME-GC-MS）, fragrance, terpene synthase, gene cloning, subcellular localization, expression analysis

孔兰, 叶秀仙, 林榕燕, 林兵, 钟淮钦. 澳洲石斛萜类合成酶基因DkTPS7的克隆与表达分析[J]. 生物技术通报, 2024, 40(12): 160-169.

KONG Lan, YE Xiu-xian, LIN Rong-yan, LIN Bing, ZHONG Huai-qin. Cloning and Expression Analysis of DkTPS7 in Dendrobium kingianum[J]. Biotechnology Bulletin, 2024, 40(12): 160-169.

图/表 8

图1 澳洲石斛花香气物质鉴定 A：花香气物质分类与比例；B：主成分分析；C：差异代谢物的韦恩图；D：K-mean分析

Fig. 1 Identification of fragrance compounds in Dendrobium kingianum A: Classification and proportion of fragrance compounds detected in flowers. B: Principal component analysis（PCA）. C: Venn diagram of differential volatile compounds. D: K-mean analysis

表1 各分组比较中差异倍数前10代谢物

Table 1 Top 10 metabolites in difference folds in each comparison of each group

分组 Groups	编号 Code	化合物 Compound	分类 Class	差异倍数 Log₂（fold change）
AZSH-B vs AZSH-F	D112	苯乙酸甲酯	酯 Ester	15.48
	XMW0856	2-羟基-1-苯基-1-丁酮	酮 Ketone	8.43
	D341	ε-衣兰油烯	萜类 Terpenoids	8.42
	D178	（1R,3aR,5aR,9aS）-1,4,4,7-四甲基-1,2,3,3a，4,5a，8,9-八氢环戊五[c]苯并呋喃	萜类 Terpenoids	7.57
	KMW0548	α-古芸烯	萜类 Terpenoids	6.46
	KMW0148	α-蒎烯	萜类 Terpenoids	6.30
	XMW0992	诺卡烯	萜类 Terpenoids	5.67
	XMW0144	（+）-樟脑	萜类 Terpenoids	5.51
	XMW1351	（3aS,8aS）-6,8a-二甲基-3-丙-2-亚基-1,2,3a,4,5,8-六氢甘菊烯	萜类 Terpenoids	5.34
	KMW0210	β-荜澄茄烯	萜类 Terpenoids	3.98
AZSH-B vs AZSH-H	D112	苯乙酸甲酯	酯 Ester	10.73
	XMW0856	2-羟基-1-苯基-1-丁酮	酮 Ketone	7.05
	D178	（1R,3aR,5aR,9aS）-1,4,4,7-四甲基-1,2,3,3a，4,5a，8,9-八氢环戊五[c]苯并呋喃	萜类 Terpenoids	5.60
	KMW0548	α-古芸烯	萜类 Terpenoids	4.95
	KMW0148	α-蒎烯	萜类 Terpenoids	4.71
	XMW0606	2-丁基-2-乙基-1,3-二氧戊环	杂环化合物Heterocyclic compound	3.24
	D305	2-甲基丁酸3-甲基丁酯	酯 Ester	3.01
	KMW0340	1-甲基庚基乙酸酯	酯 Este	3.00
	D313	（Z）-1-（1-乙氧基乙氧基）-3-己烯	醛 Aldehyd	2.96
	NMW0036	1-乙烯基-2-吡咯烷酮	酮 Ketone	2.96
AZSH-H vs AZSH-F	D341	ε-衣兰油烯	萜类 Terpenoids	8.42
	KMW0578	3-甲基丁酸芳樟酯	酯 Ester	7.75
	XMW0992	诺卡烯	萜类 Terpenoids	5.67
	D112	苯乙酸甲酯	酯 Ester	4.75
	XMW0144	（+）-樟脑	萜类 Terpenoids	3.47
	KMW0408	对伞花醇	萜类 Terpenoids	3.11
	XMW1351	（3aS,8aS）-6,8a-二甲基-3-丙-2-亚基-1,2,3a,4,5,8-六氢甘菊烯	萜类 Terpenoids	2.98
	D186	（E）-香芹醇	萜类 Terpenoids	2.39
	D401	可可醛 2	醛 Aldehyde	2.09
	XMW0149	4,5-二氢-5,5-二甲基-1H-吡唑	杂环化合物Heterocyclic compound	1.98

表1 各分组比较中差异倍数前10代谢物

Table 1 Top 10 metabolites in difference folds in each comparison of each group

分组 Groups	编号 Code	化合物 Compound	分类 Class	差异倍数 Log₂（fold change）
AZSH-B vs AZSH-F	D112	苯乙酸甲酯	酯 Ester	15.48
	XMW0856	2-羟基-1-苯基-1-丁酮	酮 Ketone	8.43
	D341	ε-衣兰油烯	萜类 Terpenoids	8.42
	D178	（1R,3aR,5aR,9aS）-1,4,4,7-四甲基-1,2,3,3a，4,5a，8,9-八氢环戊五[c]苯并呋喃	萜类 Terpenoids	7.57
	KMW0548	α-古芸烯	萜类 Terpenoids	6.46
	KMW0148	α-蒎烯	萜类 Terpenoids	6.30
	XMW0992	诺卡烯	萜类 Terpenoids	5.67
	XMW0144	（+）-樟脑	萜类 Terpenoids	5.51
	XMW1351	（3aS,8aS）-6,8a-二甲基-3-丙-2-亚基-1,2,3a,4,5,8-六氢甘菊烯	萜类 Terpenoids	5.34
	KMW0210	β-荜澄茄烯	萜类 Terpenoids	3.98
AZSH-B vs AZSH-H	D112	苯乙酸甲酯	酯 Ester	10.73
	XMW0856	2-羟基-1-苯基-1-丁酮	酮 Ketone	7.05
	D178	（1R,3aR,5aR,9aS）-1,4,4,7-四甲基-1,2,3,3a，4,5a，8,9-八氢环戊五[c]苯并呋喃	萜类 Terpenoids	5.60
	KMW0548	α-古芸烯	萜类 Terpenoids	4.95
	KMW0148	α-蒎烯	萜类 Terpenoids	4.71
	XMW0606	2-丁基-2-乙基-1,3-二氧戊环	杂环化合物Heterocyclic compound	3.24
	D305	2-甲基丁酸3-甲基丁酯	酯 Ester	3.01
	KMW0340	1-甲基庚基乙酸酯	酯 Este	3.00
	D313	（Z）-1-（1-乙氧基乙氧基）-3-己烯	醛 Aldehyd	2.96
	NMW0036	1-乙烯基-2-吡咯烷酮	酮 Ketone	2.96
AZSH-H vs AZSH-F	D341	ε-衣兰油烯	萜类 Terpenoids	8.42
	KMW0578	3-甲基丁酸芳樟酯	酯 Ester	7.75
	XMW0992	诺卡烯	萜类 Terpenoids	5.67
	D112	苯乙酸甲酯	酯 Ester	4.75
	XMW0144	（+）-樟脑	萜类 Terpenoids	3.47
	KMW0408	对伞花醇	萜类 Terpenoids	3.11
	XMW1351	（3aS,8aS）-6,8a-二甲基-3-丙-2-亚基-1,2,3a,4,5,8-六氢甘菊烯	萜类 Terpenoids	2.98
	D186	（E）-香芹醇	萜类 Terpenoids	2.39
	D401	可可醛 2	醛 Aldehyde	2.09
	XMW0149	4,5-二氢-5,5-二甲基-1H-吡唑	杂环化合物Heterocyclic compound	1.98

图2 DkTPS7蛋白生物信息学分析 A：DkTPS7蛋白的保守结构域；B：DkTPS7的亲疏水性；C：DkTPS7的二级结构（蓝色：α-螺旋，绿色：β-折角，紫色：无规则卷曲，红色：延伸链）；D：DkTPS7的三级结构

Fig. 2 Bioinformatics analysis of DkTPS7 A: Conserved domain of DkTPS7. B: Hydrophilic and hydrophobic feature of DkTPS7. C: Secondary structure of DkTPS7（Blue: α-helix, green: β-turn, purple: random coil, red: extended strand）. D: Three-dimensional structure of DkTPS7

图3 DkTPS7与其他TPS同源蛋白的多序列比对分析红色方框标注为保守基序

Fig. 3 Multi-sequence alignment of DkTPS7 with other homologous protein TPS The red boxes indicate the conserved motif

图4 DkTPS7及其他同源TPS的系统发育树

Fig. 4 Phylogenetic tree of DkTPS7 and other homologous TPSs

图5 DkTPS7亚细胞定位

Fig. 5 Subcellular localization of DkTPS7

图6 DkTPS7在不同品种（A）、不同花期（B）和不同时间点（C）中的表达模式不同小写字母表示差异显著（P< 0.05）

Fig. 6 Expression profiles of DkTPS7 at different cultivars（A）, different flowering stages（B）and different times during the blooming stage（C） Different lowercase letters indicate significant differences（P< 0.05）

图7 DkTPS7与差异代谢物的相关性 NMW0036：1-乙烯基-2-吡咯烷酮；KMW0148：α-蒎烯；XMW0606：丁基-2-乙基-1,3-二氧戊环；XMW0856：2-羟基-1-苯基-1-丁酮；KMW0340：1-甲基庚基乙酸酯；D178：（1R，3aR，5aR，9aS）-1,4,4,7-四甲基-1,2,3,3a，4,5a，8,9-八氢环戊五[c]苯并呋喃；D313：（Z）-1-（1-乙氧基乙氧基）己-3-烯；KMW0548：α-古芸烯

Fig. 7 Correlation network between DkTPS7 and differential metabolites NMW0036: 1-ethenyl-2-pyrrolidinone; KMW0148: alpha-pinene; XMW0606: 1,3-dioxolane, 2-butyl-2-ethyl; XMW0856: 1-butanone, 2-hydroxy-1-phenyl; KMW0340: 2-octanol, acetate; D178: italicene ether; D313: 3-hexene, 1-（1-ethoxyethoxy）-（Z）; KMW0548: α-gurjunene

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