SmGGPPS2对丹参酮合成的影响

doi:10.11983/CBB18222

植物学报 ›› 2019, Vol. 54 ›› Issue (2): 217-226.DOI: 10.11983/CBB18222

所属专题：逆境生物学专辑 (2019年54卷2期)

SmGGPPS2对丹参酮合成的影响

化文平^1,²,陈尘^3,⁴,智媛²,刘莉¹,王喆之²,李翠芹^2,^*()

¹ 陕西学前师范学院生命科学与食品工程学院, 西安 710061
² 陕西师范大学生命科学学院, 西北濒危药材资源开发国家工程实验室, 药用资源与天然药物化学教育部重点实验室, 西安 710062
³ 陕西省西安植物园, 西安 710068
⁴ 陕西省植物资源保护与利用工程技术研究中心, 西安 710068

收稿日期:2018-10-22 接受日期:2019-02-19 出版日期:2019-03-01 发布日期:2019-09-01
通讯作者: 李翠芹
基金资助:
国家自然科学基金(31800259);国家自然科学基金(31870276);国家重点研发计划“中医药现代化研究”专项(2018YFC1706504);陕西省教育厅科学研究基金(17JK0182);陕西省教育厅科学研究基金(2015JK1177);中央高校基本科研业务费专项资金(GK201706009);陕西省自然科学基础研究计划(2018JQ3033);陕西省自然科学基础研究计划(2018JZ3001)

Effect of SmGGPPS2 Expression on Tanshinones Biosynthesis in Salvia miltiorrhiza

Wenping Hua^1,²,Chen Chen^3,⁴,Yuan Zhi²,Li Liu¹,Zhezhi Wang²,Cuiqin Li^2,^*()

¹ Department of Life Sciences and Food Technology, Shaanxi Xueqian Normal University, Xi’an 710061, China
² Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China
³ Xi’an Botanical Garden, Xi’an 710068, China
⁴ Shaanxi Engineering Research Centre for Conservation and Utilization of Botanical Resources, Xi’an 710068, China

Received:2018-10-22 Accepted:2019-02-19 Online:2019-03-01 Published:2019-09-01
Contact: Cuiqin Li

摘要/Abstract

摘要： 香叶基香叶基焦磷酸合酶(GGPPS)是植物二萜类次生代谢物合成过程中的重要调控位点。在药用模式植物丹参(Salvia miltiorrhiza)中, GGPPS基因家族成员SmGGPPS2的生物学功能及其在丹参酮有效成分合成过程中的作用尚不明确。分别在丹参植株中过表达和RNA干涉SmGGPPS2基因, 并对转基因丹参中丹参酮含量和丹参酮合成相关基因表达量以及转基因植物生理指标进行检测, 结果表明, 过表达SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量高于野生型; RNA干涉SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量均低于野生型。调节表达SmGGPPS2后, 丹参株系中SmHMGR1和SmCPS1等多个关键酶基因的表达都呈现明显的变化。此外, 调节表达SmGGPPS2还影响丹参植株抗性。以上结果表明, SmGGPPS2在丹参酮等萜类物质的合成中起重要的调控作用。

关键词: 丹参, GGPPS, 丹参酮, 基因表达

Abstract: Geranylgerany pyrophosphate synthase (GGPPS) is an important regulator in the plant diterpenoid biosynthesis pathway. The GGPPS gene family plays a critical role in the development of the medicinal model plant Salvia miltiorrhiza. However, the biological function of SmGGPPS2, especially in the biosynthesis of tanshinone or other active ingredients, is still unclear. For functional investigation, SmGGPPS2 expression was up- or down-regulated in S. miltiorrhiza plants via overexpression or RNA interference, respectively. Then we detected the content of tanshinones, the expression of genes related to tanshinone biosynthesis, and the physiological indexes of transgenic S. miltiorrhiza plants. The content of fat-soluble components, such as tanshinone IIA and ferruginol, was increased significantly in SmGG- PPS2-overexpressed lines compared with the wild type, and the content of fat soluble-components was lower in SmGGPPS2-RNAi lines than those in the wild type lines. With the regulation of SmGGPPS2, the expression of key enzyme genes related to tanshinone biosynthesis in S. miltiorrhiza, such as SmHMGR1 and SmCPS1, was changed. In addition, the regulation of SmGGPPS2 expression also affected the resistance of S. miltiorrhiza. Our results indicate that SmGGPPS2 plays an important regulatory role in tanshinone biosynthesis.

Key words: Salvia miltiorrhiza, GGPPS, tanshinone, gene expression

化文平,陈尘,智媛,刘莉,王喆之,李翠芹. SmGGPPS2对丹参酮合成的影响. 植物学报, 2019, 54(2): 217-226.

Wenping Hua,Chen Chen,Yuan Zhi,Li Liu,Zhezhi Wang,Cuiqin Li. Effect of SmGGPPS2 Expression on Tanshinones Biosynthesis in Salvia miltiorrhiza. Chinese Bulletin of Botany, 2019, 54(2): 217-226.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18222

https://www.chinbullbotany.com/CN/Y2019/V54/I2/217

图/表 8

表1 本研究中载体构建及检测所用引物

Table 1 Primers used for vector construction and detection in this study

Primer name	Sequence (5′→3′)
OG2S (KpnI)	GGGGTACCGCGAAGAGCGTGGAAGC- AGA
OG2R (BamHI)	CGGGATCC GGCAATAGCAATCAAGGGAGCA
G2i1S (EcoRI)	CGGAATTC CTCCTCTCAACCTCTGTCAAAACTC
G2i1R (KpnI)	GGGGTACCTCATCTGGACCACTGCC- TCC
G2i2S (BamHI)	CGGGATCC CTCCTCTCAACCTCTGTCAAAACTC
G2i2R (HindIII)	CCCAAGCTTTCATCTGGACCACTGCC- TCC
35SF	GCCGTGAAGACTGGCGAACA
35SR	AGGAAGGGTCTTGCGAAGGATAGT

表2 本研究中qPCR检测所用引物

Table 2 Primers used for qPCR in this study

Primer name	Sequence (5′→3′)	Primer name	Sequence (5′→3′)
ActinS	AGGAACCACCGATCCAGACA	SmDXS1S	TGAGAGCGACTACGACTGCTTTGG
ActinR	GGTGCCCTGAGGTCCTGTT	SmDXS1R	CCCATCCAGATTGGCAGTAGGC
SmHMGR1S	GCAACCATCTACTCTCGTCCCA	SmDXS2S	GGTCGAGGAACTGGAGGGATTG
SmHMGR1R	GTGCTCCATGAGCTGCATCAG	SmDXS2R	CGTCAGGATTTCGTGCGGATA
SmHMGR2S	GGGTTCAACTACGAGGCCATACTG	SmDXS3S	CACGAATGGGCTGCCAAAAT
SmHMGR2R	TGTTTGTGCTCGCCACCAGG	SmDXS3R	CCATCGAATCCAATGAAGCCAC
SmHMGR3S	AGTCTCGTGATGTCCCTGCTCG	SmGGPPS1S	GGGGCTATTTTGGGAGGTGGAA
SmHMGR3R	GCCTCAACCTGCTTGGCGTA	SmGGPPS1R	CAGCAGCTTGGGATACGTGGTC
SmIPI1S	AGCGTGCATCCAAATCCAGAC	SmGGPPS2S	CGGTCTCCTCTCAACCTCTGTCAA
SmIPI1R	GATAGCTTCAAGCCCCCCTCA	SmGGPPS2R	CTCCTTCATCTGGACCACTGCCT
SmCPS1S	ATGGATGGGCAGCAGCAGTAAA	SmGGPPS3S	GGCCAGTGCTCTGCTGTCTGTG
SmCPS1R	CGTCCTCAACAACGTCCTGATGTATT	SmGGPPS3R	TCGGCCACCTCCATCGCTT

图1 丹参转基因株系及其DNA水平的PCR检测 (A) SmGGPPS2过表达代表性株系(OG2-3)、SmGGPPS2 RNA干涉表达代表性株系(IG2-6)及野生型对照(Bar=2 cm); (B) SmGGPPS2过表达转基因株系(1-13); (C) SmGGPPS2- RNAi转基因株系(1-20)。M: DNA标准量DL2000; WT: 野生型; c: 阴性对照; p: 阳性对照

Figure 1 Phenotypes and PCR-screening of the transgenic lines of Salvia miltiorrhiza (A) SmGGPPS2-overexpression transgenic line (OG2-3), SmGGPPS2-RNAi transgenic line (IG2-6), and wild type (Bar= 2 cm); (B) SmGGPPS2-overexpression transgenic lines (1-13); (B) SmGGPPS2-RNAi transgenic lines (1-20). M: DNA marker DL2000; WT: Wild type; c: Negative control; p: Positive control

图2 SmGGPPS2在丹参过表达(A)和RNA干涉(B)株系中的表达WT: 野生型。* 差异显著(P?0.05); ** 差异显著(P?0.01); *** 差异显著(P? 0.001)。数据均进行归一化处理。

Figure 2 Expression of SmGGPPS2 in overexpression (A) and RNAi (B) Salvia miltiorrhiza transgenic linesWT: Wild type. * Significant differences (P?0.05); ** Significant differences (P?0.01); *** Significant differences (P? 0.001). The data are normalized.

图3 GC-MS检测转基因丹参提取物总离子流色谱峰A: 邻苯二甲酸二异丁酯; B: 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione; C: 铁锈醇; D: 单棕榈酸甘油; E: 邻苯二甲酸二异辛酯; F: 羟基陶塔酚; G: 丹参酮IIA; H: 隐丹参酮; I: 菜油甾醇; J: 豆甾醇; K: β-谷甾醇

Figure 3 GC-MS chromatogram of Salvia miltiorrhiza transgenic linesA: Phthalic acid diisobutyl ester; B: 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione; C: Ferruginol; D: b-monopalmitin; E: Diisooctyl phthalate; F: 7β-hydroxytotarol; G: Tanshinone IIA; H: Cryptotanshinone; I: Campesterol; J: Stigmasterol; K: β- sitosterol

图4 转基因丹参株系脂溶性提取物中不同成分含量比较(A)及不同转基因丹参株系中丹参酮IIA含量(B) A–K同图3。WT: 野生型。*** 差异显著(P<0.001)

Figure 4 Contents of different ingredients from fat-soluble extract in transgenic lines (A) and contents oftanshinone IIA in different transgenic lines (B) of Salvia miltiorrhizaA–K see Figure 3. WT: Wild type. *** Significant differences (P<0.001)

图5 转基因丹参株系中丹参酮合成途径相关基因的表达水平WT: 野生型。* 差异显著(P<0.05); ** 差异显著(P<0.01); *** 差异显著(P<0.001)

Figure 5 Relative expression levels of genes involved in tanshinones biosynthesis of Salvia miltiorrhiza transgenic lines WT: Wild type. * Significant differences (P<0.05); ** Significant differences (P<0.01); *** Significant differences (P<0.001)

图6 不同丹参转基因株系抗逆生理指标SOD: 超氧化物歧化酶; MDA: 丙二醛; POD: 过氧化物酶; WT: 野生型。* 差异显著(P<0.05); ** 差异显著(P<0.01)

Figure 6 The resistance physiological indexes of different Salvia miltiorrhiza transgenic linesSOD: Superoxide dismutase; MDA: Malondialdehyde; POD: Peroxidase; WT: Wild type. * Significant differences (P<0.05); ** Significant differences (P<0.01)

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SmGGPPS2对丹参酮合成的影响

Effect of SmGGPPS2 Expression on Tanshinones Biosynthesis in Salvia miltiorrhiza

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