Cloning and Expression Analysis of DkTPS7 in Dendrobium kingianum

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

Abstract

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

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.

Figures/Tables 8

References 28

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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

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