艾龙脑脱氢酶基因AArBDH1的克隆及功能鉴定

doi:10.11983/CBB22123

摘要/Abstract

摘要： 龙脑是艾(Artemisia argyi)叶中最重要的药效成分之一, 具有抗菌、消炎及镇痛等药理活性。其生物合成和代谢受多种酶的影响, 其中龙脑脱氢酶是将龙脑氧化为樟脑的关键酶之一。利用气相色谱-质谱联用(GC-MS)技术测定了35份艾种质叶片中龙脑和樟脑的含量。结果表明, 不同品种艾叶片中龙脑和樟脑含量差异较大, 在部分种质中樟脑含量甚至超过了龙脑, 说明大量的龙脑被氧化为樟脑后严重降低了艾叶中龙脑的含量。基于全长转录组和同源性比较分析, 在艾叶中克隆到1个艾龙脑脱氢酶编码基因AArBDH1。AArBDH1基因含有2个外显子和1个内含子, 编码包含289个氨基酸残基的蛋白。qRT-PCR分析表明, 在艾不同组织和不同发育时期的叶片中AArBDH1呈差异表达, 在茎和30天叶龄的叶片中高表达。以龙脑为底物、NAD⁺为辅酶的酶促反应表明, AArBDH1能催化龙脑脱氢生成樟脑。该研究为进一步揭示艾叶片中龙脑积累的调控和改进机制提供了理论依据和基因资源。

关键词: 艾, 龙脑, 龙脑脱氢酶, 樟脑

Abstract: Borneol is one of the most important pharmacodynamic components in Artemisia argyi, which has antibacterial, anti-inflammatory, analgesic and other pharmacological activities. The synthesis and metabolism of borneol are affected by many kinds of enzymes, and borneol dehydrogenase is one of the key enzymes that oxidize borneol to camphor. In this study, the contents of borneol and camphor in 35 germplasm of A. argyi leaves were determined by Gas chromatograph-mass spectrometer (GC-MS). We found that the amount of borneol and camphor was highly varied among the varieties, and some germplasm contained more camphor than borneol, indicating that a large proportion of borneol was oxidized to camphor in A. argyi leaves. Based on the full-length transcriptome of A. argyi and homology comparison analysis, we cloned the first borneol dehydrogenase encoding gene AArBDH1 in A. argyi. The AArBDH1 contained 2 exons and 1 intron, and encoded a protein of 289 amino acids. The AArBDH1 gene expression levels were measured by real-time quantitative reverse transcription PCR (qRT-PCR) technology, which showed that AArBDH1 was differentially expressed in different tissues and in leaves at different development stages, and highly expressed in stem and 30 days old leaves. The enzymatic reactions of AArBDH1 with borneol as substrate and NAD⁺ as coenzyme showed that AArBDH1 could catalyze the dehydrogenation of borneol to camphor. Our study provides a theoretical basis and gene resource for further analyzing the regulation and potential improvement of borneol accumulation in A. argyi leaves.

Key words: Artemisia argyi, borneol, borneol dehydrogenase, camphor

陈昌婕, 苗玉焕, 罗丹丹, 王梓欣, 郭璐娟, 赵婷婷, 刘大会. 艾龙脑脱氢酶基因AArBDH1的克隆及功能鉴定. 植物学报, 2023, 58(4): 560-572.

Changjie Chen, Yuhuan Miao, Dandan Luo, Zixin Wang, Lujuan Guo, Tingting Zhao, Dahui Liu. Cloning and Functional Verification of the Borneol Dehydrogenase Encoding Gene AArBDH1 in Artemisia argyi. Chinese Bulletin of Botany, 2023, 58(4): 560-572.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22123

https://www.chinbullbotany.com/CN/Y2023/V58/I4/560

图/表 11

图1 艾叶挥发油GS-MS总离子色谱图 1: 樟脑; 2: 龙脑

Figure 1 The total ion chromatogram of volatile oil in Artemisia argyi leaves by GC-MS 1: Camphor; 2: Borneol

图2 35份艾种质叶片中龙脑和樟脑的相对含量 S01-S35为35份艾叶样品编号。

Figure 2 Relative contents of borneol and camphor in the leaves of 35 Artemisia argyi varieties S01-S35 is the number of 35 Artemisia argyi varieties.

图3 AArBDH1与AaBDH、ADH2、LiBDH、CcBDH1、CcBDH2和CcBDH3的多序列比对

Figure 3 Multiple sequence alignment of AArBDH1 with AaBDH, ADH2, LiBDH, CcBDH1, CcBDH2, and CcBDH3

图4 AArBDH1和21种SDR蛋白的系统进化树

Figure 4 Phylogenetic tree of AArBDH1 and other 21 SDR proteins

表1 从NCBI下载的SDR蛋白质编码基因信息

Table 1 Information of SDR encoding genes downloaded from NCBI

No.	Gene	Species	GenBank accession	Functional annotation	Reference
1	AaBDH	Artemisia annua	KT070864	Borneol dehydrogenase	Tian et al., 2015
2	ADH2	A. annua	GU253890	Alcohol dehydrogenase	Polichuk et al., 2010
3	ALDH	Pseudomonas aeruginosa	WP_028626884	Dehydrogenase	Tsang et al., 2016
4	CcBDH1	Cinnamomum camphora	MW012431	Borneol dehydrogenase	Ma et al., 2021a
5	CcBDH2	C. camphora	MW012432	Borneol dehydrogenase	Ma et al., 2021a
6	CcBDH3	C. camphora	MN551096	Borneol dehydrogenase	Ma et al., 2021a
7	CHCR	Cricetulus griseus	AB020238	Carbonyl reductase	Terada et al., 2000
8	DlSDR	Digitalis lanata	Q93Y47	3-beta-hydroxysteroid dehydrogenase	Finsterbusch et al., 1999
9	FiSDR	Forsythia intermedia	AF352735	Secoisolariciresinol dehydrogenase	Xia et al., 2001
10	ISPD	Mentha × piperita	AAU20370	(-)-isopiperitenol dehydrogenase	Ringer et al., 2005
11	HSCR	Homo sapiens	J04056	Carbonyl reductase	Wermuths et al., 1988
12	LiBDH	Lavandula × intermedia	AFV30207	Borneol dehydrogenase	Sarker et al., 2012
13	MIR	Mentha × piperita	AY300162	(-)-isopiperitenone reductase	Ringer et al., 2003
14	MNR	Mentha × piperita	ABC88670	(+)-neomenthol reductase	Davis et al., 2005
15	MMR	Mentha × piperita	AAQ55960	Menthol dehydrogenase	Davis et al., 2005
16	NEPS	Nepeta mussinii	6F9Q_D	Nepetalactol-related short-chain dehydrogenase	Lichman et al., 2019
17	PsSDR	Pisum sativum	AAF04253	Short-chain alcohol dehydrogenase	Brosché and Strid, 1999
18	PTCR	Sus scrofa	M80709	20-beta-hydroxysteroid dehydrogenase	Tanaka et al., 1992
19	RED1	A. annua	GU167953	Reductase/dehydrogenase	Rydén et al., 2010
20	SalR	Papaver bracteatum	EF184229	Salutaridine reductase	Geissle et al., 2007
21	ZSD1	Zingiber zerumbet	AB480831	Short-chain dehydrogenase	Okamoto et al., 2011

表1 从NCBI下载的SDR蛋白质编码基因信息

Table 1 Information of SDR encoding genes downloaded from NCBI

No.	Gene	Species	GenBank accession	Functional annotation	Reference
1	AaBDH	Artemisia annua	KT070864	Borneol dehydrogenase	Tian et al., 2015
2	ADH2	A. annua	GU253890	Alcohol dehydrogenase	Polichuk et al., 2010
3	ALDH	Pseudomonas aeruginosa	WP_028626884	Dehydrogenase	Tsang et al., 2016
4	CcBDH1	Cinnamomum camphora	MW012431	Borneol dehydrogenase	Ma et al., 2021a
5	CcBDH2	C. camphora	MW012432	Borneol dehydrogenase	Ma et al., 2021a
6	CcBDH3	C. camphora	MN551096	Borneol dehydrogenase	Ma et al., 2021a
7	CHCR	Cricetulus griseus	AB020238	Carbonyl reductase	Terada et al., 2000
8	DlSDR	Digitalis lanata	Q93Y47	3-beta-hydroxysteroid dehydrogenase	Finsterbusch et al., 1999
9	FiSDR	Forsythia intermedia	AF352735	Secoisolariciresinol dehydrogenase	Xia et al., 2001
10	ISPD	Mentha × piperita	AAU20370	(-)-isopiperitenol dehydrogenase	Ringer et al., 2005
11	HSCR	Homo sapiens	J04056	Carbonyl reductase	Wermuths et al., 1988
12	LiBDH	Lavandula × intermedia	AFV30207	Borneol dehydrogenase	Sarker et al., 2012
13	MIR	Mentha × piperita	AY300162	(-)-isopiperitenone reductase	Ringer et al., 2003
14	MNR	Mentha × piperita	ABC88670	(+)-neomenthol reductase	Davis et al., 2005
15	MMR	Mentha × piperita	AAQ55960	Menthol dehydrogenase	Davis et al., 2005
16	NEPS	Nepeta mussinii	6F9Q_D	Nepetalactol-related short-chain dehydrogenase	Lichman et al., 2019
17	PsSDR	Pisum sativum	AAF04253	Short-chain alcohol dehydrogenase	Brosché and Strid, 1999
18	PTCR	Sus scrofa	M80709	20-beta-hydroxysteroid dehydrogenase	Tanaka et al., 1992
19	RED1	A. annua	GU167953	Reductase/dehydrogenase	Rydén et al., 2010
20	SalR	Papaver bracteatum	EF184229	Salutaridine reductase	Geissle et al., 2007
21	ZSD1	Zingiber zerumbet	AB480831	Short-chain dehydrogenase	Okamoto et al., 2011

图5 AArBDH1基因的结构(A)、在不同组织中的表达丰度(B)和AArBDH1蛋白质结构分析(C) UTR: 非翻译区; CDS: 编码区。Leaf A、Leaf B、Leaf C、Leaf D分别为叶芽及叶龄为15天、30天和45天的叶片。不同小写字母表示差异显著(P<0.05)。

Figure 5 Gene structure (A), expression profiles in different organizations of AArBDH1 (B) and protein structure analysis of AArBDH1 (C) UTR: Untranslated region; CDS: Coding sequence. Leaf A, Leaf B, Leaf C and Leaf D represent leaf buds, 15 days, 30 days and 45 days old leaves, respectively. Different lowercase letters indicate significant differences (P<0.05).

图6 AArBDH1蛋白质结构预测 (A) AArBDH1的亚细胞定位预测; (B) AArBDH1蛋白质二级结构预测; (C) AArBDH1蛋白质三级结构预测

Figure 6 Protein structure prediction of AArBDH1 (A) The subcellular localization prediction of AArBDH1; (B) The secondary structure prediction of AArBDH1; (C) The tertiary structure prediction of AArBDH1

图7 AArBDH1的克隆(A)和阳性克隆鉴定(B) M: 分子标记; 编号为待检测样品的序号。

Figure 7 Cloning of AArBDH1 (A) and the identification of positive clones (B) M: Markers; Numbers: The number of the samples tested.

图8 AArBDH1的诱导表达产物和纯化 1: 未经诱导的菌体蛋白; 2: IPTG诱导表达的菌体蛋白; 3: 1次纯化后的菌体蛋白; 4: 2次纯化后的菌体蛋白

Figure 8 Induced expression product and purification of AA- rBDH1 1: Expression products without IPTG; 2: Induced expression by IPTG; 3: Primary purified protein; 4: Secondary purified protein

图9 龙脑的GC-MS分析 (A) 色谱峰; (B) 碎片离子峰

Figure 9 GC-MS analysis of borneol (A) Chromatographic peak; (B) Fragment ion peak

图10 AArBDH1催化产物的GC-MS分析 CG: 对照组; E1, E2: 实验组; A: 色谱峰; B: 碎片离子峰

Figure 10 GC-MS analysis of products catalyzed by AArBDH1 CG: Control group; E1, E2: Experimental groups; A: Chromatographic peak; B: Fragment ion peak

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