PjFPS和Pjβ-AS双基因协同作用对珠子参皂苷生物合成的影响

doi:10.3969/j.issn.1004-1524.2022.03.02

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (3): 428-436.DOI: 10.3969/j.issn.1004-1524.2022.03.02

PjFPS和Pjβ-AS双基因协同作用对珠子参皂苷生物合成的影响

陈勤¹^,²(), 刘美佳¹^,², 刘迪秋¹^,², 曲媛¹^,², 崔秀明¹^,², 葛锋¹^,²^,^*()

1.昆明理工大学生命科学与技术学院,云南昆明 650500
2.云南省三七资源可持续利用重点实验室,云南昆明 650500

收稿日期:2021-07-26 出版日期:2022-03-25 发布日期:2022-03-30
通讯作者: 葛锋
作者简介:*葛锋,E-mail: panaxcordyceps@163.com
陈勤(1995—),男,云南曲靖人,硕士研究生,研究方向为药用植物生物技术。E-mail: 2213537688@qq.com
基金资助:
云南省基础研究计划重点项目(202101AS070024);云南省重大科技计划(2019ZF011-2);国家自然科学基金(82060692)

Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins

CHEN Qin¹^,²(), LIU Meijia¹^,², LIU Diqiu¹^,², QU Yuan¹^,², CUI Xiuming¹^,², GE Feng¹^,²^,^*()

1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
2. Key Laboratory of Panax Notoginseng Resources Sustainable Development and Utilization,Kunming 650500, China

Received:2021-07-26 Online:2022-03-25 Published:2022-03-30
Contact: GE Feng

摘要/Abstract

摘要：

法尼基焦磷酸合酶(PjFPS)和β-香树脂醇合酶(Pjβ-AS)是珠子参皂苷(Panax japonicus saponins,PJS)生物合成途径中可能的关键酶。通过在珠子参(P.japonicus)细胞中同时过表达PjFPS和Pjβ-AS基因,探讨PjFPS和Pjβ-AS对PJS生物合成的协同作用。结果表明,PjFPS和Pjβ-AS是PJS生物合成途径中的关键酶基因,对珠子参皂苷的生物合成具有调节作用。过表达PjFPS和Pjβ-AS的细胞系中,PJS合成途径相关的多个关键酶基因的表达水平有不同程度的提高,且PJS的含量最高为普通细胞系的2.4倍。虽然单独过表达PjFPS也可增加PJS的合成,但双基因(PjFPS和Pjβ-AS)协同调控PJS合成的效果更好。

关键词: 三萜皂苷, 生物合成, 珠子参, 法尼基焦磷酸合酶, β-香树脂醇合酶

Abstract:

Farnesyl pyrophosphate synthase (PjFPS) and β-amyrin synthase (Pjβ-AS)are key enzymes in the biosynthetic pathway of Panax japonicus saponins (PJS). In this study, effects of PjFPS and PjβAS on PJS biosynthesis were investigated by overexpressing PjFPS and Pjβ-AS simultaneously in P. japonicus cells. The results showed that PjFPS and Pjβ-AS were key enzyme genes in PJS biosynthetic pathway and had regulatory effects on biosynthesis of P. japonicus. The cell lines overexpressing PjFPS and Pjβ-AS showed differential increases in the expression levels of several key enzyme genes related to the PJS synthesis pathway, and the highest levels of PJS were up to 2.4 times higher than those of the normal cell lines. Although overexpression of PjFPS alone also increased PJS synthesis, dual genes (PjFPS and Pjβ-AS) synergistically regulated PJS synthesis more effectively.

Key words: triterpenoid saponins, biosynthesis, Panax japonicus, farnesyl pyrophosphate synthase, β-amyrin synthase

中图分类号:

S567.5

陈勤, 刘美佳, 刘迪秋, 曲媛, 崔秀明, 葛锋. PjFPS和Pjβ-AS双基因协同作用对珠子参皂苷生物合成的影响[J]. 浙江农业学报, 2022, 34(3): 428-436.

CHEN Qin, LIU Meijia, LIU Diqiu, QU Yuan, CUI Xiuming, GE Feng. Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 428-436.

图/表 7

表1 PCR引物

Table 1 Primers used for PCR

基因	上游引物Forward primer(5'→3')	下游引物Reverse primer (5'→3')	产物长度Product length/bp
Pjβ-AS	ATGTGGAGGCTAATGACAGCCAAGGG	TCAGACGCTTTTAGGTGGTAATCGAACA	2 384
PjFPS	ACAGACAACAACTTCCCCTCCAT	AGAATGAGCGATCTGAAGACGAG	1 136
nptⅡ	CTCTGATGCCGCCGTGTT	CCCTGATGCTCTTCGTCCA	450
hptⅡ	GAAGTGCTTGACATTGGGGAAT	AGATGTTGGCGACCTCGTATT	450

表2 qRT-PCR引物

Table 2 Primers used for qRT-PCR

基因	上游引物Forward primer (5'→3')	下游引物Reverse primer (5'→3')	产物长度Product length/bp
Pjβ-AS	ATGTGGAGGCTAATGACAGCCAAGGG	TCAGACGCTTTTAGGTGGTAATCGAACA	174
PjFPS	ACAGACAACAACTTCCCCTCCAT	AGAATGAGCGATCTGAAGACGAG	168
18S RNA	AACCATAAACGATGCCGACCAG	TTCAGCCTTGCGACCATACTCC	162
PjDS	TGGGAGTTTCAGCCCGATG	GGGGAGGTGTATAAAGTAAAGAGCC	141
PjHMGR	GACATTTTGAACACCCTTGGACA	CTCTTAATTTTGATACACTGGCCGT	172
PjSE	TAGGTGAACTTCTACAACCAGGAGG	CAACCAGAGATGTAACAGTCCCC	178
PjSS	CGAGCACTTGACACTGTTGAGGAT	CTATTGCCTCCTGGTAACCGTTTC	185

图1 转基因珠子参细胞中nptⅡ和hptⅡ的PCR分析 A, nptⅡ基因的PCR结果;B, hptⅡ基因的PCR结果;M,DL2000 DNA marker;1,对照组细胞;2,携带hptⅡ基因空载的转基因细胞系;3~6,PjFPS/β-AS转基因细胞。

Fig.1 PCR analysis of nptⅡ and hpt Ⅱ in transgenic P. japonicus cells A, PCR result of nptⅡ; B, PCR result of hptⅡ; M, DL2000 DNA marker; 1, Cell lines of control; 2, Transgenic cell lines containing empty vector of hptⅡ gene; 3-6, PjFPS/β-AS-transgenic cells.

图2 转PjFPS/β-AS珠子参细胞关键酶基因的相对表达水平 FPS,法尼基焦磷酸合成酶基因;SS,鲨烯合酶基因;β-AS,β-香树脂醇合成酶基因;HMGR,3-羟基-3-甲基戊二酸单酰辅酶A还原酶基因;SE,鲨烯环氧化酶基因;DS,达玛烯二醇合成酶基因。WT,对照组细胞系;T1、T2、T3和T4,PjFPS/β-AS过表达细胞系;T5,转空载体转基因细胞系。*和**分别表示与对照组相比在P<0.05和P<0.01水平差异显著。下同。

Fig.2 Relative expression levels of key enzyme genes inP. japonicus cells FPS, Farnesyl pyrophosphate synthase gene; SS, squalene synthase gene; β-AS, β-amyrin synthase gene; HMGR, 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene; SE, squalene cyclooxygenase gene; DS, damacendiol synthase gene. WT, wild-type cell line; T1, T2, T3 and T4, four PjFPS/β-AS overexpression cell lines; T5, trans-empty vector transgenic cell line. * and ** meant significant differences between control (non-transgenic cells) and transgenic cell lines at the level of P<0.05 andP<0.01. The same as below.

图3 珠子参细胞系中FPS和β-AS活性 WT,对照组细胞系;F1、F2、F3和F4,PjFPS转基因细胞系;F5,转空载体细胞系。下同。

Fig.3 Activities of FPS and β-AS in P. japonicus cell lines WT, Wild-type cell line; F1, F2, F3 and F4, PjFPS transgenic cell lines; F5, Trans-empty vector cell lines.The same as below.

图4 过表达皂苷合成关键酶基因珠子参细胞的总皂苷含量

Fig.4 Content of total saponins in transgenic P. japonicus cells

图5 珠子参细胞系中单体皂苷的含量

Fig.5 Contents of monomer saponins in P. japonicus cell lines

参考文献 26

[1]	王丽, 苏钛, 侯安国. 珠子参的化学成分及药理作用研究进展[J]. 中国中医基础医学杂志, 2020(7): 1037-1040.
	WANG L, SU T, HOU A G. Research progress on chemical constituents and pharmacological effects of Panax japonicus[J]. Journal of Basic Chinese Medicine, 2020(7): 1037-1040. (in Chinese with English abstract)
[2]	KOCHKIN D V, KACHALA V V, SHASHKOV A S, et al. Malonyl-ginsenoside content of a cell-suspension culture of Panax japonicus var. Repens[J]. Phytochemistry, 2013, 93: 18-26. DOI URL
[3]	QI L W, WANG C Z, YUAN C S. Ginsenosides from American ginseng: chemical and pharmacological diversity[J]. Phytochemistry, 2011, 72(8): 689-699. DOI URL
[4]	LUO H M, SUN C, SUN Y Z, et al. Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers[J]. BMC Genomics, 2011, 12(Suppl 5): S5.
[5]	欧阳丽娜, 向大位, 吴雪, 等. 竹节参化学成分及药理活性研究进展[J]. 中草药, 2010, 41(6): 1023-1027.
	OUYANG L N, XIANG D W, WU X, et al. Research progress on the chemical constituents and pharmacological activities of Panax japonicus[J]. Acupuncture Research, 2010, 41(6): 1023-1027. (in Chinese)
[6]	DENG B, ZHANG P, GE F, et al. Enhancement of triterpenoid saponins biosynthesis in Panax notoginseng cells by co-overexpressions of 3-hydroxy-3-methylglutaryl CoA reductase and squalene synthase genes[J]. Biochemical Engineering Journal, 2017, 122: 38-46. DOI URL
[7]	ZHANG D H, JIANG L X, LI N, et al. Overexpression of the squalene epoxidase gene alone and in combination with the 3-hydroxy-3-methylglutaryl coenzyme A gene increases ganoderic acid production in Ganoderma Lingzhi[J]. Journal of Agricultural and Food Chemistry, 2017, 65(23): 4683-4690. DOI URL
[8]	刘美佳, 于怡琳, 姜森, 等. 珠子参中法尼基焦磷酸合酶(FPS)对皂苷生物合成的影响研究[J]. 植物研究, 2018(4):611-618.
	LIU M J, YU Y L, JIANG S, et al. Effect of farnesyl-pyrophosphate synthase(FPS) on the biosynthesis of saponins in Panax japonicus[J]. Bulletin of Botanical Research, 2018(4):611-618. (in Chinese with English abstract)
[9]	WANG J, LI Y, LIU D. Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poriacocos[J]. International Journal of Molecular Sciences, 2014, 15(12): 22188-22202. DOI URL
[10]	杨延, 张翔, 姜森, 等. 珠子参中皂苷成分及其药理活性研究进展[J]. 食品工业科技, 2019, 40(2): 347-356.
	YANG Y, ZHANG X, JIANG S, et al. Research progress on saponins and pharmacological activities of Panax japonicus[J]. Science and Technology of Food Industry, 2019, 40(2): 347-356. (in Chinese)
[11]	HUANG Z W, LIN J C, CHENG Z X, et al. Production of oleanane-type sapogenin in transgenic rice via expression of β-amyrin synthase gene from Panax japonicus C. A. Mey[J]. BMC Biotechnology, 2015, 15: 45. DOI URL
[12]	ZHAO C, XU T H, LIANG Y L, et al. Functional analysis of β-amyrin synthase gene in ginsenoside biosynthesis by RNA interference[J]. Plant Cell Reports, 2015, 34(8): 1307-1315. DOI URL
[13]	DENG B, HUANG Z J, GE F, et al. An AP2/ERF family transcription factor PnERF1 raised the biosynthesis of saponins in Panax notoginseng[J]. Journal of Plant Growth Regulation, 2017, 36(3): 691-701. DOI URL
[14]	HOFGEN R, WILLMITZER L. Storage of competent cells for Agrobacterium transformation[J]. Nucleic Acids Research, 1988, 16(20): 9877. DOI URL
[15]	ZHANG X, GE F, DENG B, et al. Molecular cloning and characterization of PnbHLH 1 transcription factor in Panax notoginseng[J]. Molecules (Basel, Switzerland), 2017, 22(8): 1268. DOI URL
[16]	ABOUL-MAATY N A F, ORABY H A S. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method[J]. Bulletin of the National Research Centre, 2019, 43: 25. DOI URL
[17]	平洁, 梁赅, 汪晖, 等. 一种检测HMG-CoA还原酶活性的改良分光光度法及应用:CN102221531A[P]. 2011-10-19.
[18]	李敏. 珠子参化学成分及生物活性研究[D]. 长春: 吉林大学, 2017.
	LI M. Studies on the chemical constituents and bioactivities of Panacis majoris Rhizoma[D]. Changchun: Jilin University, 2017. (in Chinese with English abstract)
[19]	ZHANG S P, WANG G, ZUO T, et al. Comparative transcriptome analysis of rhizome nodes and internodes in Panax japonicus var. major reveals candidate genes involved in the biosynthesis of triterpenoid saponins[J]. Genomics, 2020, 112(2): 1112-1119. DOI URL
[20]	TANG Q Y, CHEN G, SONG W L, et al. Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides[J]. Planta, 2019, 249(2): 393-406. DOI URL
[21]	陈勤, 雷君, 刘迪秋, 等. 人参属三萜皂苷骨架修饰的研究进展[J]. 中药材, 2020, 43(11): 2830-2836.
	CHEN Q, LEI J, LIU D Q, et al. The research progress of ginseng triterpenoid saponin skeleton modifying enzyme[J]. Journal of Chinese Medicinal Materials, 2020, 43(11): 2830-2836. (in Chinese)
[22]	YANG Y, GE F, SUN Y, et al. Strengthening triterpene saponins biosynthesis by over-expression of farnesyl pyrophosphate synthase gene and RNA interference of cycloartenol synthase gene in Panax notoginseng cells[J]. Molecules (Basel, Switzerland), 2017, 22(4): 581. DOI URL
[23]	KIM Y K, KIM Y B, UDDIN M R, et al. Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase[J]. ACS Synthetic Biology, 2014, 3(10): 773-779. DOI URL
[24]	LIU Y, CHEN H H, WEN H, et al. Enhancing the accumulation of beta-amyrin in Saccharomyces cerevisiae by co-expression of Glycyrrhiza uralensis squalene synthase 1 and beta-amyrin synthase genes[J]. Acta Pharmaceutica Sinica, 2014, 49(5): 734-741.
[25]	LU J, LI J X, WANG S H, et al. Advances in ginsenoside biosynthesis and metabolic regulation[J]. Biotechnology and Applied Biochemistry, 2018, 65(4): 514-522. DOI URL
[26]	ZHAO C, XU T H, LIANG Y L, et al. Functional analysis of β-amyrin synthase gene in ginsenoside biosynthesis by RNA interference[J]. Plant Cell Reports, 2015, 34(8): 1307-1315. DOI URL

PjFPS和Pjβ-AS双基因协同作用对珠子参皂苷生物合成的影响

Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins

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