Cloning and Functional Verification of the Borneol Dehydrogenase Encoding Gene AArBDH1 in Artemisia argyi

doi:10.11983/CBB22123

Abstract

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

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[J]. Chinese Bulletin of Botany, 2023, 58(4): 560-572.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22123

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

Figures/Tables 11

References 43

[1]	陈昌婕, 罗丹丹, 苗玉焕, 康利平, 郭兰萍, 刘大会, 黄璐琦 (2021). 艾种质资源挥发性成分分析与评价. 中国中药杂志 46, 3814-3823.
[2]	陈昌婕, 马琳, 康利平, 苗玉焕, 陈中文, 郭兰萍, 刘大会, 黄璐琦 (2020). 不同种植时期和垄作模式对蕲艾生长和品质的影响. 中国中药杂志 45, 4041-4050.
[3]	丁贤华, 张丽萍, 费璇, 朱强, 张金华, 任仙樱, 吴志刚, 姜程曦 (2021). 温郁金法呢基焦磷酸合酶(CwFPPS)基因的克隆、亚细胞定位及表达分析. 中草药 52, 6968-6974.
[4]	国家药典委员会 (2020). 中华人民共和国药典. 北京: 中国医药科技出版社. pp. 92.
[5]	兰晓燕, 张元, 朱龙波, 刘大会, 黄显章, 周利, 康利平 (2020). 艾叶化学成分、药理作用及质量研究进展. 中国中药杂志 45, 4017-4030.
[6]	刘星星, 张茜, 郭夏丽, 龚上佶, 江香梅, 付宇新, 罗丽萍 (2014). 不同化学型樟树叶挥发性成分组成的多变量分析. 植物学报 49, 161-166. DOI
[7]	马琳, 陈昌婕, 康利平, 苗玉焕, 方艳, 郭兰萍, 刘大会, 黄璐琦 (2020). 不同种植密度、叶位与叶龄对蕲艾产量和品质的影响. 中国中药杂志 45, 4031-4040.
[8]	马青, 马蕊, 靳保龙, 崔光红 (2021). 天然冰片资源研究进展. 中国中药杂志 46, 57-61.
[9]	田梅, 陈灵丽, 靳保龙, 郭娟, 葛慧, 赵鑫, 崔光红 (2021). 铁棒锤转录组分析及乌头碱生物合成相关基因的挖掘. 药学学报 56, 3353-3361.
[10]	王文然, 张文颖, 管乐, 汤崴, 房经贵, 王晨 (2017). 石榴木质素相关蛋白PgCAD蛋白的生物信息学分析. 果树学报 34, 67-75.
[11]	徐应文, 吕季娟, 吴卫, 郑有良 (2009). 植物单萜合酶研究进展. 生态学报 29, 3188-3197.
[12]	张雪琳, 陈新旺, 吴毅明 (2021). 近10年来艾叶挥发油的化学成分及药理活性研究进展. 中华中医药学刊 39(5), 111-118.
[13]	赵圆圆, 孙叶雯, 郑诗敏, 李萌, 马东明, 杨锦芬 (2022). 阳春砂龙脑基二磷酸合酶关键氨基酸位点筛选及突变体的构建. 中草药 53, 529-537.
[14]	郑昆, 钟肖飞, 张华 (2020). 艾叶挥发油类成分及其药理作用的研究进展. 中国实验方剂学杂志 26(18), 224-234.
[15]	Amstutz CL, Fristedt R, Schultink A, Merchant SS, Niyogi KK, Malnoë A (2020). An atypical short-chain dehydrogenase-reductase functions in the relaxation of photo- protective qH in Arabidopsis. Nat Plants 6, 154-166. DOI
[16]	Brosché M, Strid Ǻ (1999). Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses. Plant Physiol 121, 479-488. DOI PMID
[17]	Davis EM, Ringer KL, McConkey ME, Croteau R (2005). Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint. Plant Physiol 137, 873-881. DOI PMID
[18]	Dong TW, Chen N, Ma X, Wang J, Wen J, Xie Q, Ma R (2018). The protective roles of L-borneolum, D-borneolum and synthetic borneol in cerebral ischaemia via modulation of the neurovascular unit. Biomed Pharmacother 102, 874-883. DOI URL
[19]	Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C, Luckner M (1999). Δ⁵-3β-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. — a multifunctional enzyme in steroid metabolism? Planta 209, 478-486. PMID
[20]	Geissler R, Brandt W, Ziegler J (2007). Molecular modeling and site-directed mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/ reductase salutaridine reductase. Plant Physiol 143, 1493-1503. PMID
[21]	Ho TJ, Hung CC, Shih TL, Yiin LM, Chen HP (2018). Investigation of borneols sold in Taiwan by chiral gas chromatography. J Food Drug Anal 26, 348-352. DOI URL
[22]	Hurd MC, Kwon M, Ro DK (2017). Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif. Biochem Biophys Res Commun 490, 963-968. DOI URL
[23]	Khine AA, Lu PC, Ko TP, Huang KF, Chen HP (2020). Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1. Protein Expression Purif 175, 105715. DOI URL
[24]	Lichman BR, Kamileen MO, Titchiner GR, Saalbach G, Stevenson CEM, Lawson DM, O’Connor SE (2019). Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis. Nat Chem Biol 15, 71-79. DOI PMID
[25]	Lu DD, Tan ZW, Li L, Yu YL, Xu LJ, Yang HQ, Dong W, Liang HZ (2022). Cloning and expression analysis of anthocyanidin reductase gene ANR in Carthamus tinctorius L. J Nucl Agric Sci 36, 517-526.
[26]	Ma R, Su P, Guo J, Jin BL, Ma Q, Zhang HY, Chen LL, Mao LY, Tian M, Lai CJS, Tang JF, Cui GH, Huang LQ (2021a). Bornyl diphosphate synthase from Cinnamomum burmanni and its application for (+)-borneol biosynthesis in yeast. Front Bioeng Biotechnol 9, 631863. DOI URL
[27]	Ma R, Su P, Jin BL, Guo J, Tian M, Mao LY, Tang JF, Chen T, Lai CJS, Zeng W, Cui GH, Huang LQ (2021b). Molecular cloning and functional identification of a high- efficiency (+)-borneol dehydrogenase from Cinnamomum camphora (L.) Presl. Plant Physiol Biochem 158, 363-371. DOI URL
[28]	Miao YH, Luo DD, Zhao TT, Du HZ, Liu ZH, Xu ZP, Guo LP, Chen CJ, Peng SN, Li JX, Ma L, Ning GG, Liu DH, Huang LQ (2022). Genome sequencing reveals chromosome fusion and extensive expansion of genes related to secondary metabolism in Artemisia argyi. Plant Biotechnol J 20, 1902-1915. DOI URL
[29]	Okamoto S, Yu FN, Harada H, Okajima T, Hattan JI, Misawa N, Utsumi R (2011). A short-chain dehydrogenase involved in terpene metabolism from Zingiber zerumbet. FEBS J 278, 2892-2900. DOI PMID
[30]	Polichuk DR, Zhang YS, Reed DW, Schmidt JF, Covello PS (2010). A glandular trichome-specific monoterpene alcohol dehydrogenase from Artemisia annua. Phytochemistry 71, 1264-1269. DOI PMID
[31]	Ringer KL, Davis EM, Croteau R (2005). Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint. Plant Physiol 137, 863-872. DOI PMID
[32]	Ringer KL, McConkey ME, Davis EM, Rushing GW, Croteau R (2003). Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint. Arch Biochem Biophys 418, 80-92. DOI PMID
[33]	Rydén AM, Ruyter-Spira C, Litjens R, Takahashi S, Quax W, Osada H, Bouwmeester H, Kayser O (2010). Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua. Plant Cell Physiol 51, 1219-1228. DOI URL
[34]	Sarker LS, Galata M, Demissie ZA, Mahmoud SS (2012). Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula × intermedia. Arch Biochem Biophys 528, 163-170. DOI URL
[35]	Tanaka M, Ohno S, Adachi S, Nakajin S, Shinoda M, Nagahama Y (1992). Pig testicular 20 beta-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity. cDNA cloning of pig testicular 20 beta- hydroxysteroid dehydrogenase. J Biol Chem 267, 13451-13455. PMID
[36]	Terada T, Sugihara Y, Nakamura K, Sato R, Inazu N, Maeda M (2000). Cloning and bacterial expression of monomeric short-chain dehydrogenase/reductase (carbonyl reductase) from CHO-K1 cells. Eur J Biochem 267, 6849-6857. PMID
[37]	Tian N, Tang YW, Xiong S, Tian DM, Chen YH, Wu DC, Liu ZH, Liu SQ (2015). Molecular cloning and functional identification of a novel borneol dehydrogenase from Artemisia annua L. Ind Crops Prod 77, 190-195. DOI URL
[38]	Tsang HL, Huang JL, Lin YH, Huang KF, Lu PL, Lin GH, Khine AA, Hu A, Chen HP (2016). Borneol dehydrogenase from Pseudomonas sp. strain TCU-HL1 catalyzes the oxidation of (+)-borneol and its isomers to camphor. Appl Environ Microbiol 82, 6378-6385. DOI URL
[39]	Wang H, Ma DM, Yang JF, Deng K, Li M, Ji XY, Zhong LT, Zhao HY (2018). An integrative volatile terpenoid profiling and transcriptomics analysis for gene mining and functional characterization of AvBPPS and AvPS involved in the monoterpenoid biosynthesis in Amomum villosum. Front Plant Sci 9, 846. DOI URL
[40]	Wermuth B, Bohren KM, Heinemann G, von Wartburg JP, Gabbay KH (1988). Human carbonyl reductase. Nucleotide sequence analysis of a cDNA and amino acid sequence of the encoded protein. J Biol Chem 263, 16185-16188. PMID
[41]	Xia ZQ, Costa MA, Pélissier HC, Davin LB, Lewis NG (2001). Secoisolariciresinol dehydrogenase purification, cloning, and functional expression. Implications for human health protection. J Biol Chem 276, 12614-12623. DOI PMID
[42]	Youn B, Moinuddin SGA, Davin LB, Lewis NG, Kang C (2005). Crystal structures of Apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans. J Biol Chem 280, 12917-12926. DOI URL
[43]	Yu CS, Lin CJ, Hwang JK (2004). Predicting subcellular localization of proteins for Gram-negative bacteria by sup- port vector machines based on n-peptide compositions. Protein Sci 13, 1402-1406. DOI URL

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

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