药学学报

Primer name	Primer sequence (5′-3′)	Fragment length /bp
Screening primers of overexpression hairy roots
SmHPPR1-F1	ttggatccaccactcaaacaccatct	1 019
SmHPPR1-R1	tgaagctttcaaagagttatcaaac
SmHPPR1-F2	tcaggatccatggaggcgatcggtgttc	933
SmHPPR1-R2	cagtctagaaggtgttaacagaggct
Kan-F	cacaacagacaatcggc	660
Kan-R	cgtaaagcacgaggaag
qPCR primes
qActin-F	aggaaccaccgatccagaca	278
qActin-R	ggtgccctgaggtcctgtt
qSmHPPR1-F	cgccgacgccgatatcatcgactca	190
qSmHPPR1-R	aatccgcctcagaaccgccagcat

Primer name	Primer sequence (5′-3′)	Fragment length /bp
Screening primers of overexpression hairy roots
SmHPPR1-F1	ttggatccaccactcaaacaccatct	1 019
SmHPPR1-R1	tgaagctttcaaagagttatcaaac
SmHPPR1-F2	tcaggatccatggaggcgatcggtgttc	933
SmHPPR1-R2	cagtctagaaggtgttaacagaggct
Kan-F	cacaacagacaatcggc	660
Kan-R	cgtaaagcacgaggaag
qPCR primes
qActin-F	aggaaccaccgatccagaca	278
qActin-R	ggtgccctgaggtcctgtt
qSmHPPR1-F	cgccgacgccgatatcatcgactca	190
qSmHPPR1-R	aatccgcctcagaaccgccagcat

Sample	Sampling time	Dry weight/g	Salvianolic acid/mg·g^-1 DW	Lignin/%
Control-3	2010	0.56	/	45.45	34.51	79.96	/
2022	0.96 ± 0.11	1.71 ± 0.37	67.42 ± 3.70	23.46 ± 4.20	90.88 ± 7.45	11.38 ± 0.37
SmHPPR1-OE-3	2008	0.61	/	60.28	22.64	82.92	/
2022	0.73 ± 0.01	0.62 ± 0.07	73.65 ± 4.85	30.51 ± 2.78	104.16 ± 4.28	12.70 ± 1.36

Sample	Sampling time	Dry weight/g	Salvianolic acid/mg·g^-1 DW	Lignin/%
Control-3	2010	0.56	/	45.45	34.51	79.96	/
2022	0.96 ± 0.11	1.71 ± 0.37	67.42 ± 3.70	23.46 ± 4.20	90.88 ± 7.45	11.38 ± 0.37
SmHPPR1-OE-3	2008	0.61	/	60.28	22.64	82.92	/
2022	0.73 ± 0.01	0.62 ± 0.07	73.65 ± 4.85	30.51 ± 2.78	104.16 ± 4.28	12.70 ± 1.36

Sample	Raw_read	Clean_read	Error/%	Q20/%	Q30/%	GC/%
Control-3-①	82 142 460	80 974 024	0.02	98.18	94.45	48.10
Control-3-②	71 304 730	70 535 362	0.02	98.20	94.50	48.22
SmHPPR1-OE-3-①	75 248 990	74 743 574	0.02	98.24	94.58	47.39
SmHPPR1-OE-3-②	74 884 956	74 421 244	0.02	98.43	95.1	47.37

Sample	Raw_read	Clean_read	Error/%	Q20/%	Q30/%	GC/%
Control-3-①	82 142 460	80 974 024	0.02	98.18	94.45	48.10
Control-3-②	71 304 730	70 535 362	0.02	98.20	94.50	48.22
SmHPPR1-OE-3-①	75 248 990	74 743 574	0.02	98.24	94.58	47.39
SmHPPR1-OE-3-②	74 884 956	74 421 244	0.02	98.43	95.1	47.37

Gene	Gene_id	log₂FC	NR_annotation
SmTAT2	TR739_c4_g1	1.05	Tyrosine aminotransferase 2 [Salvia miltiorrhiza]
Sm4CL4	TR174_c0_g1	-1.28	4-Coumarate: coenzyme A ligase 4 [Salvia miltiorrhiza]
Sm4CL6	TR4603_c0_g1	-2.17	4-Coumarate: coenzyme A ligase 6, partial [Salvia miltiorrhiza]
SmRAS4-like	TR5466_c0_g1	-3.00	Rosmarinic acid synthase 4, partial [Salvia miltiorrhiza]
Sm4CL9	TR8805_c0_g1	-3.53	4-Coumarate--CoA ligase 9[Salvia miltiorrhiza]
SmRAS6-like1	TR2028_c4_g1	-4.08	Rosmarinic acid synthase 6 [Salvia miltiorrhiza]
SmRAS6-like2	TR35281_c0_g1	-6.49	Rosmarinic acid synthase 6 [Salvia miltiorrhiza]

Gene	Gene_id	log₂FC	NR_annotation
SmTAT2	TR739_c4_g1	1.05	Tyrosine aminotransferase 2 [Salvia miltiorrhiza]
Sm4CL4	TR174_c0_g1	-1.28	4-Coumarate: coenzyme A ligase 4 [Salvia miltiorrhiza]
Sm4CL6	TR4603_c0_g1	-2.17	4-Coumarate: coenzyme A ligase 6, partial [Salvia miltiorrhiza]
SmRAS4-like	TR5466_c0_g1	-3.00	Rosmarinic acid synthase 4, partial [Salvia miltiorrhiza]
Sm4CL9	TR8805_c0_g1	-3.53	4-Coumarate--CoA ligase 9[Salvia miltiorrhiza]
SmRAS6-like1	TR2028_c4_g1	-4.08	Rosmarinic acid synthase 6 [Salvia miltiorrhiza]
SmRAS6-like2	TR35281_c0_g1	-6.49	Rosmarinic acid synthase 6 [Salvia miltiorrhiza]

Gene	Gene_id	log₂FC	NR_annotation
CYT	TR74706_c0_g1	-1.59	Cytochrome b6-f complex iron-sulfur subunit [Salvia splendens]
NADP+	TR26565_c0_g1	-1.92	Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) [Salvia splendens]
NADP+	TR68771_c0_g1	-2.91	Glyceraldehyde-3-phosphate dehydrogenase (NADP+) [Salvia splendens]

Gene	Gene_id	log₂FC	NR_annotation
CYT	TR74706_c0_g1	-1.59	Cytochrome b6-f complex iron-sulfur subunit [Salvia splendens]
NADP+	TR26565_c0_g1	-1.92	Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) [Salvia splendens]
NADP+	TR68771_c0_g1	-2.91	Glyceraldehyde-3-phosphate dehydrogenase (NADP+) [Salvia splendens]

丹参SmHPPR1基因调控丹酚酸生物合成的研究

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谈荣慧 , 赵望 , 张金家 , 赵淑娟 ^*

药学学报 | 研究论文 2023,58(9): 2818-2828

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药学学报 |研究论文 2023 , 58 (9) : 2818 -2828

丹参SmHPPR1基因调控丹酚酸生物合成的研究

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谈荣慧, 赵望, 张金家, 赵淑娟^*

作者信息

上海中医药大学中药研究所, 中药新资源与品质评价国家中医药管理局重点研究室, 中药标准化教育部重点实验室, 上海市复方中药重点实验室, 上海 201203

通讯作者:

*赵淑娟, Tel: 86-21-51322576, E-mail: zhaoshujuan@126.com; zhaoshujuan@shutcm.edu.cn

SmHPPR1 from Salvia miltiorrhiza regulated the biosynthesis of salvianolic acids

Rong-hui TAN, Wang ZHAO, Jin-jia ZHANG, Shu-juan ZHAO^*

Affiliations

The SATCM Key Laboratory for New Resources & Quality Evaluation of Chinese Medicine, The MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

出版时间: 2023-09-12 doi: 10.16438/j.0513-4870.2023-0030

文章导航

摘要

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丹参是临床上治疗心脑血管疾病的常用中药, 其主要水溶性活性成分为迷迭香酸和丹酚酸B, 由苯丙烷类代谢途径产生。4-羟基苯丙酮酸还原酶(HPPR) 是苯丙烷代谢途径中的关键酶。本课题组前期在丹参中克隆了SmHPPR1基因并构建了植物表达载体。在此基础上, 获得了携带SmHPPR1的转基因拟南芥阳性植株并测定4-羟基苯乳酸的含量; 诱导了过表达SmHPPR1丹参毛状根并进行有效成分含量测定和转录组分析。结果显示: 携带SmHPPR1的T1代转基因拟南芥中4-羟基苯乳酸含量为0.594 mg·g^-1; SmHPPR1-OE-3毛状根中的迷迭香酸、丹酚酸B、总丹酚酸含量分别是control-3毛状根的1.09、1.29、1.15倍, 丹参素是control-3毛状根的36.26%; 转录组分析显示, SmHPPR1过表达引起了SmTAT2表达量上调; 蛋白-蛋白相互作用表明CYT (TR74706_c0_g1)、NADP⁺ (TR26565_c0_g1) 和NADP⁺ (TR68771_c0_g1) 是网络的中心节点并参与代谢过程、细胞过程等。以上研究表明, SmHPPR1在携带SmHPPR1的转基因拟南芥中能催化4-羟基苯丙酮酸还原为4-羟基苯乳酸; 丹参毛状根中过表达SmHPPR1可以通过协同调控其他途径基因来提高丹酚酸的含量。

关键词

丹参 / 4-羟基苯丙酮酸还原酶 / 丹酚酸 / 迷迭香酸 / 生物合成

Abstract

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Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb widely used to treat cardiovascular and cerebrovascular diseases at clinic. Its main water-soluble components are rosmarinic acid (RA) and salvianolic acid B (SAB), which are produced by phenylpropanoid pathway. 4-Hydroxyphenylpyruvate reductase (HPPR) is a key enzyme in phenylpropanoid metabolism pathway. SmHPPR1 was cloned from S. miltiorrhiza and was constructed into plant expression vector pJR-SmHPPR1. On this basis, SmHPPR1 transgenic Arabidopsis plants were induced and the content of 4-hydroxyphenyllactic acid (pHPL) was determined. SmHPPR1-overexpressing (SmHPPR1-OE) hairy roots of S. miltiorrhiza were obtained and the concentration of active components and transcriptome analysis were performed. The results showed that the concentration of pHPL in SmHPPR1 transgenic Arabidopsis T1 was 0.594 mg·g^-1 dry weight. The concentration of RA, SAB and total salvianolic acid in SmHPPR1-OE-3 hairy roots were 1.09, 1.29, 1.15 times of that in control-3, respectively, and the content of Danshensu was 36.26% of that in control-3. Transcriptomic analysis revealed that overexpression of SmHPPR1 caused the upregulation of other phenylpropanoid pathway genes like SmTAT2. Protein-protein interaction indicated CYT (TR74706_c0_g1), NADP⁺ (TR26565_c0_g1) and NADP⁺ (TR68771_c0_g1) is the central node of the network and participated in metabolic process and cellular process. The tracking work in this study proved that SmHPPR1 could catalyze the reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid in SmHPPR1 transgenic Arabidopsis, and SmHPPR1-overexpressing in hairy roots of S. miltiorrhiza could increase the concentration of salvianolic acids through synergistically regulating other pathway genes.

Key words

Salvia miltiorrhiza Bunge / 4-hydroxyphenylpyruvate reductase / salvianolic acid / rosmarinic acid / biosynthesis

引用本文

谈荣慧, 赵望, 张金家, 赵淑娟. 丹参SmHPPR1基因调控丹酚酸生物合成的研究. 药学学报, 2023 , 58 (9) : 2818 -2828 . DOI: 10.16438/j.0513-4870.2023-0030

Rong-hui TAN, Wang ZHAO, Jin-jia ZHANG, Shu-juan ZHAO. SmHPPR1 from Salvia miltiorrhiza regulated the biosynthesis of salvianolic acids[J]. Acta Pharmaceutica Sinica, 2023 , 58 (9) : 2818 -2828 . DOI: 10.16438/j.0513-4870.2023-0030

正文

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丹参(Salvia Miltiorrhiza Radix et Rhizoma) 是唇形科鼠尾草属植物丹参(Salvia miltiorrhiza Bunge) 的干燥根和根茎, 具有活血祛瘀, 调经止痛、清心除烦、凉血消痈的功效^[1]。其基原植物具有生命力强、周期短、基因组小、染色体数目少等特点, 随着组织培养和转基因技术成熟、丹参转录组和基因组信息的逐步丰富, 丹参被认为是中药研究的理想模式植物^{[2, 3]}。丹参中的有效成分主要分为两类: 脂溶性的二萜醌类化合物, 如丹参酮I、丹参酮IIA、隐丹参酮等; 和水溶性的酚酸类成分(丹酚酸类化合物), 如丹酚酸B (salvianolic acid B, SAB)、丹酚酸A、迷迭香酸(rosmarinic acid, RA) 和丹参素(danshensu, DSU) 等。研究表明, 丹参水溶性成分具有抗心肌缺血、抗血栓、抗高血脂、抗胃溃疡、调节组织修复和再生等作用^[4-6]。

丹酚酸类化合物由苯丙氨酸和酪氨酸两条相互平行的分支途径产生。苯丙氨酸被苯丙氨酸解氨酶(PAL)、肉桂酸4-羟化酶(C4H) 和4-香豆酸辅酶A连接酶(4CL) 催化产生4-香豆酰CoA; 酪氨酸在酪氨酸转氨酶(TAT) 作用下生成4-羟苯基丙酮酸(pHPP), pHPP被4-羟基苯丙酮酸还原酶(HPPR) 催化为4-羟基苯乳酸(pHPL)。活化形式的4-香豆酰CoA和pHPL在迷迭香酸合成酶(RAS) 和细胞色素P450蛋白—CYP98A14的催化下生成迷迭香酸^[7-10] (图 1)。同时, 4-香豆酰CoA在一系列木质素合成酶作用下生成木质素^{[11, 12]}。

HPPR广泛分布于陆生植物中, 在苔藓、蕨类植物、裸子植物和被子植物中均发现了与HPPR结构同源^[13]。近年来, 多种植物中克隆鉴定出HPPR基因, 如彩叶草(Coleus blumei)^[14]、洋苏草(Salvia offificinalis)^[15]、黄芩(Scutellaria baicalensis)^[16]、紫苏(Perilla frutescens)^[17]、猫须草(Orthosiphon aristatus)^[18]、拟南芥(Arabidopsis thaliana)^[13]、小麦(Triticum aestivuml)^[19]等。研究发现, HPPR属于D-异构体特异性2-羟基酸脱氢酶家族^[20]。大肠杆菌中异源表达的CbHPPR可催化NAD(P)H依赖的4-羟基苯丙酮酸还原为4-羟基苯乳酸, 3, 4-二羟基苯丙酮酸还原为3, 4-二羟基苯乳酸^[14]。紫苏PfHPPR在根、茎、叶中均有表达, 但在叶片中表达量最高, 并受脱落酸(ABA)、水杨酸(SA)、紫外线-B辐射(UV-B) 等多种因素诱导^[17]。小麦TaHPPR在根、小穗、叶鞘中均有表达, 且在低温、干旱、ABA和高盐胁迫处理下表达水平均有所下降^[19]。

本课题组前期实验中克隆到丹参SmHPPR1 (GenBank: DQ266514), 编码313aa, 并构建植物表达载体pJR-SmHPPR1。本研究在此基础上, 将植物表达载体分别导入根癌农杆菌GV3101和发根农杆菌LBA9402, 侵染拟南芥幼苗和丹参无菌苗幼嫩叶片诱导携带SmHPPR1的转基因拟南芥和过表达SmHPPR1丹参毛状根; 筛选携带SmHPPR1的转基因拟南芥并测定pHPL的含量; 筛选过表达SmHPPR1丹参毛状根并进行有效成分含量测定和转录组分析, 以此来探讨SmHPPR1在丹酚酸合成途径中的作用, 为利用基因工程手段合成丹酚酸有效成分奠定基础。

材料与方法

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材料丹参植株(两年生) 采自上海中医药大学百草园, 收集盛花期丹参植株的根、叶和花组织材料, 液氮速冻、并于-80 ℃冰箱保存备用。丹参无菌苗(MS固体培养基培养) 由本实验室培养。所用大肠杆菌Top10、pJR质粒、pJR-SmHPPR1质粒、发根农杆菌LBA9402均为本实验室保存。拟南芥种植于20 ℃植物培养箱, 光周期为16 h/8 h (亮/暗)。

试剂和仪器 PrimeScript^TM RT reagent Kit反转录试剂盒、SYBR Green^® premix Ex Taq^TM试剂盒(大连宝生物工程有限公司); PR3聚合酶、DNA Marker (上海创英生物科技公司); 总RNA提取试剂盒(上海捷瑞生物工程有限公司); 木质素含量检测试剂盒、引物合成(上海生工生物工程有限公司); RA和SAB对照品(上海融禾医药科技发展有限公司); 乙腈(色谱纯, Fisher公司), 其余试剂为分析纯。实时荧光定量PCR仪-StepOnePlus (ABI); Agilent 1100和1260高效液相色谱仪; BioTek Synergy H1酶标仪, Percival培养箱。

植物表达载体的构建 将实验室保种的T-SmHPPR1和pJR质粒分别用BamH I和Sal I核酸内切酶双酶切, 胶回收目的片段后连接并转化大肠杆菌DH5α感受态细胞, PCR扩增筛选阳性单菌落, 将单菌落摇菌提质粒酶切鉴定并送测序, 经鉴定正确的重组质粒命名为pJR-SmHPPR1。

根癌农杆菌介导的遗传转化 利用冻融法将pJR-SmHPPR1质粒转化根癌农杆菌GV3101, 将PCR验证正确的阳性单克隆命名为GV3101-SmHPPR1并转接于20 mL含有50 mg·L^-1庆大霉素(gentamycin, Gm) 和50 mg·L^-1卡那霉素(kanamycin, Kan) 的LB液体培养基中, 28 ℃、220 r·min^-1培养过夜; 取培养物, 加入200 mL含有50 mg·L^-1 Kan的LB液体培养基中, 28 ℃、220 r·min^-1培养至A₆₀₀ = 0.6~1.0; 将培养物于5 000 r·min^-1, 4 ℃离心15 min, 收集菌体, 用MSOH液体培养基重悬, 使A₆₀₀ = 0.6~1.0备用。MSOH是在MS培养基的基础上由1 g·L^-1水解酪蛋白替代NH₄NO₃^[21]。

转基因拟南芥株系的获得及筛选 转化前4天将已抽苔的花序剪掉, 选取生长良好的植物并使土壤吸足水。把GV3101-SmHPPR1农杆菌悬浮液倒在搪瓷盘中, 将长有植物的培养杯在其中倒置, 保证植株莲座叶以上部分浸没于液体中, 15 s后取出转化植株, 平放在一个铁盘中, 盖上保鲜膜, 移入温室暗培养, 第二天揭去保鲜膜, 竖直培养。转化的拟南芥生长3到4周后, 收种子并放在干燥环境中存放2周。将经过消毒的种子铺种于含有50 mg·L^-1 Kan的MS培养基上, 春化处理, 移至温室6~8天后, 挑出能正常生长的绿苗, 移栽到土壤中。用CTAB法^[22]提取植株基因组DNA并用引物SmHPPR1-F1、SmHPPR1-R1扩增SmHPPR1基因条带, 筛选携带SmHPPR1的转基因拟南芥阳性植株。

转基因拟南芥植株酚酸类物质含量的测定 样品溶液的制备: 收取成熟刚收集种子的野生拟南芥(WT)、携带SmHPPR1的转基因拟南芥植株, 冷冻干燥后剪成2~4 mm的小段, 分别精密称取100.00 mg后转移至三角瓶中, 加入蒸馏水15 mL, 暗处放置18 h (期间可轻轻摇晃), 取上清液适量, 微孔滤膜滤过后, 即得样品溶液1和2。标准品溶液的制备: 精密称取pHPP适量于容量瓶中, 加甲醇溶解并定容, 得对照品浓度为2.44 mg·mL^-1; 精密称取pHPL适量于量瓶中, 加甲醇溶解并定容, 得标准品浓度为2.14 mg·mL^-1。内参样品制备: 精密吸取pHPL标准品50 μL于两个5 mL量瓶中, 分别用样品溶液1和2稀释定容, 得样品溶液3和4。色谱条件: Agilent 1100高效液相色谱仪, 色谱柱为C18 (4.6 mm × 250 mm, 5 μm), 柱温25 ℃, 流速1.0 mL·min^-1, 进样量20 μL, 检测波长280 nm, 流动相为乙腈(A) 和0.4%甲酸(B) 梯度洗脱: 0~40 min, A的比例由0到30%, B的比例由100%到70%。

发根农杆菌介导的遗传转化 利用三亲杂交法将pJR-SmHPPR1质粒和pJR质粒转入发根农杆菌LBA9402, PCR验证正确的阳性单克隆命名为LBA9402-SmHPPR1和LBA9402-control, 并转接于3 mL含有50 mg·L^-1的利福平(rifampicin, Rif) 和50 mg·L^-1 Kan的YMB液体培养基中, 28 ℃、200 r·min^-1震荡培养至YMB液体培养基变为浑浊的淡黄色, 按1%比例接种至20 mL YMB液体培养基中, 28 ℃、200 r·min^-1震荡培养至A₆₀₀为0.5备用。

丹参毛状根的诱导及筛选 参照文献^[21]的实验方法诱导丹参毛状根。将活化的LBA9402-SmHPPR1和LBA9402-control的菌液稀释2倍, 侵染丹参无菌苗幼嫩叶片, 5 min后放在MSOH固体培养基上25 ℃暗培养; 2天后用无菌水洗去叶片表面的菌液并放在含有500 mg·L^-1头孢噻肟(cefotaxime sodium, Cef) 的MSOH固体培养基上25 ℃暗培养; 2周左右在叶片周围长出毛状根, 待毛状根生长到2 cm左右时剪下并转移到含有500 mg·L^-1 Cef和50 mg·L^-1 Kan的MSOH固体培养基中培养; 3周后将毛状根转移到含有250 mg·L^-1 Cef的MSOH固体培养基中培养; 2~3周后转移到MSOH液体培养基中培养。脱菌完全的丹参毛状根在MSOH液体培养基上继代培养, 每21天转接一次。采用CTAB法^[22]提取毛状根基因组DNA并分别用引物SmHPPR1-F2、SmHPPR1-R2和Kan-F、Kan-R (表 1) 扩增SmHPPR1和Kan基因条带, 筛选过表达SmHPPR1和载体对照毛状根株系并命名为: SmHPPR1-OE和control。

丹参毛状根丹酚酸的提取测定 以“1 g鲜重/100 mL MSOH液体培养基”接种丹参毛状根, 21天后收取丹参毛状根材料并于-80 ℃冰箱中冷冻, 冷冻干燥机冻干并研磨成粉, 储存于电子干燥器中备用。

RA和SAB的提取参照文献^[23]的实验方法, 精密称取粉末50 mg, 加入70%甲醇和1%甲酸10 mL, 常温浸泡30 min, 超声40 min。

DSU的提取精密称取粉末100 mg, 加入70%甲醇和1%甲酸5 mL, 37 ℃摇床振摇30 min, 60 ℃超声60 min, 4 000 r·min^-1离心5 min, 取上清。沉淀加入70%甲醇和1%甲酸5 mL并重复上述操作, 将两者上清合并并用N₂挥干, 加入70%甲醇和1%甲酸1 mL超声30 min。

标准品溶液的制备分别精密称取DSU、RA和SAB对照品, 加入70%甲醇和1%甲酸制成DSU、RA和SAB标准品溶液。

色谱条件测定RA和SAB的色谱条件(2008年、2010年) 同测定转SmHPPR1拟南芥植株中酚酸类物质含量的方法; 测定RA和SAB的色谱条件(2022年): 与上述不同的是采用Agilent 1260高效液相色谱仪, 检测波长286和330 nm, 流动相A (25%) 和B (75%) 在0~30 min等比例洗脱。测定DSU的色谱条件: 与测定RA和SAB色谱条件(2022年) 不同的是, 检测波长300 nm, 乙腈(A) 和0.4%甲酸(B) 梯度洗脱: 0~10 min, A 5%, B 95%; 11~20 min, A 25%, B 75%; 21~40 min, A 90%, B 10%; 41~45 min, A 5%, B 95%。

木质素含量测定 精密称取5 mg丹参毛状根粉末于1.5 mL离心管中, 按照木质素含量检测试剂盒说明书操作, 酶标仪分别测定280 nm下样品管和空白管的吸光值(A)。

实时荧光定量PCR分析(qRT-PCR) 采用Primer Premier 5.0软件设计qPCR引物(表 1), 对引物的扩增效率进行了评估, qSmHPPR1和qActin的引物扩增效率范围为95%~105%。提取SmHPPR1-OE-3和control-3毛状根的总RNA, 根据反转录试剂盒说明书合成cDNA第一链, -20 ℃冰箱保存备用。将反转录得到的cDNA稀释5倍, 按照SYBR Green^®Premix Ex TaqTM试剂盒说明书加样并进行qPCR反应, 以丹参Actin基因为内参, 用2^-ΔΔCt方法计算相对表达水平^[24]。

转录组测序及生物信息学分析 将过表达丹参毛状根SmHPPR1-OE-3和载体对照丹参毛状根control-3送到上海中科新生命公司进行cDNA文库构建和转录组测序。去除接头序列和低质量的reads, 获得可用于后续分析的clean reads并由Trinity软件进行转录本拼接。应用DESeq2软件进行样品组间的差异表达分析, 对原有假设检验得到的P-value进行校正, 以差异表达倍数(fold change, FC) 和错误发现率(false discovery rate, FDR) 作为差异表达基因(differentially expressed genes, DEGs) 的筛选条件, 筛选标准为|log₂FC| > 1且FDR < 0.05。利用KEGG数据库预测DEGs主要参与的代谢途径和信号通路。利用HemI 1.0软件绘制各样本中基因表达量的热图。利用STRING数据库(https://www.string-db.org/) 预测SmHPPR1-OE-3毛状根的DEGs对应的蛋白之间可能存在的相互作用(protein-protein interaction, PPI) 并用Cytoscape 3.9.1软件构建预测的PPI网络。

结果与分析

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1 丹参SmHPPR1的聚类分析

为了研究丹参SmHPPRs家族成员的亲缘关系, 基于SmHPPR1、SmHPPR2 (GenBank: KF220565)、SmHPPR3 (GenBank: KF220566) 和SmHPPR4 (GenBank: KF220566) 蛋白的氨基酸, 同时选择拟南芥(Arabidopsis thaliana) 和彩叶草(Coleus blumei) 的HPPR蛋白序列为参照, 利用MEGA 7.0软件、使用邻接法(neighbor-joining) 中重复1 000次抽样的方法构建系统进化树。聚类结果(图 2) 显示, SmHPPR1与彩叶草(CbHPPR) 亲缘关系最近, 其次是拟南芥(AtHPPR2、AtHPPR3)。

2 转基因拟南芥植株的获得和筛选

冻融法将pJR-SmHPPR1质粒转入根癌农杆菌GV3101, 再以浸泡法转化拟南芥。T0代拟南芥种子经表面消毒后, 平铺于含50 mg·L^-1 Kan的MS培养基上(每个培养皿约300粒种子)。从大约600粒种子中得到10棵能正常发芽生长的植株(图 3A)。结果表明: 不含Kan抗性的植株子叶变黄, 不能继续生长, 而含Kan抗性的植株长势良好。

将抗性筛选后的小苗移栽到土壤中, 待长出较大的真叶后, 提取叶片的基因组DNA, 以WT拟南芥植株为对照, 用SmHPPR1-F1/SmHPPR1-R1为引物PCR扩增, 发现绝大部分Kan抗性植株的PCR结果都是阳性的(图 3B)。可以确定这些拟南芥植株已转入丹参SmHPPR1基因。

3 转基因拟南芥酚酸类物质含量测定

HPLC法检测WT拟南芥和携带SmHPPR1的转基因拟南芥中HPPR催化的产物—4-羟基苯乳酸的含量。如图所示, 野生型拟南芥中未检测到4-羟基苯乳酸, 转SmHPPR1 T1代拟南芥中4-羟基苯乳酸含量为0.594 mg·g^-1干重(dry weight, DW) (图 4)。推测SmHPPR1基因在拟南芥中的表达产物可能参与了4-羟基苯乳酸的生物合成反应。

4 SmHPPR1过表达丹参毛状根的获得和筛选

LBA9402-SmHPPR1菌液侵染丹参无菌苗叶片, 3周后叶片周围长出2 cm左右的丹参毛状根(图 5A), 将毛状根剪下转移到含有500 mg·L^-1 Cef和50 mg·L^-1 Kan的MSOH固体培养基中培养(图 5B), 经过多次转接直至脱菌完成, 将毛状根转接到MSOH液体培养基中暗培养(图 5C)。LBA9402-control诱导丹参毛状根的过程与上述过程类似。提取丹参毛状根基因组DNA, PCR扩增SmHPPR1和Kan目的条带(图 6A、B), PCR条带与目的条带大小一致, 说明诱导的丹参毛状根过表达4个株系和载体对照4个株系均为阳性。根据毛状根生长状态和丹酚酸含量选取SmHPPR1-OE-3和control-3作为后期研究对象, 其中SmHPPR1-OE-3毛状根中的RA、SAB和总丹酚酸(total salvianolic acids, TSA, RA和SAB之和) 含量分别是60.28、22.64和82.92 mg·g^-1; control-3毛状根中的RA、SAB和TSA含量分别是45.45、34.51和79.96 mg·g^-1; SmHPPR1-OE-3和control-3毛状根的干重分别是0.61和0.56 g (表 2)。

5 培养丹参毛状根中丹酚酸含量的测定

继代培养后, 再次考察SmHPPR1-OE-3和control-3毛状根的丹酚酸含量。收取以“1 g鲜重/100 mL MSOH液体培养基”中培养21天的SmHPPR1-OE-3和control-3丹参毛状根材料(图 5D、E) 并冷冻干燥研磨成粉, 高效液相测定SmHPPR1-OE-3和control-3毛状根中DSU、RA和SAB含量, SmHPPR1-OE-3毛状根中的DSU、RA、SAB和TSA含量分别是0.62 ± 0.07、73.65 ± 4.85、30.51 ± 2.78和104.16 ± 4.28 mg·g^-1; control-3毛状根中的DSU、RA、SAB和TSA含量分别是1.71 ± 0.37、67.42 ± 3.70、23.46 ± 4.20和90.88 ± 7.45 mg·g^-1; SmHPPR1-OE-3和control-3毛状根的干重分别是0.73 ± 0.01和0.96 ± 0.11 g (表 2)。SmHPPR1-OE-3毛状根中的RA、SAB、TSA含量分别是control-3毛状根的1.09、1.29、1.15倍, DSU是control-3毛状根的36.26%。SPSS软件分析表明SmHPPR1-OE-3和control-3毛状根中RA、SAB和TSA含量无显著差异, DSU和干重有显著差异。

6 丹参毛状根木质素含量的测定

SmHPPR1-OE-3和control-3毛状根木质素含量分别是(11.38 ± 0.37)%、(12.70 ± 1.36)% (表 2)。SPSS软件分析表明SmHPPR1-OE-3和control-3毛状根中木质素含量无显著差异。

7 测序数据分析和KEGG富集分析

为了研究SmHPPR1基因对丹参毛状根中丹酚酸含量的影响, 将SmHPPR1-OE-3和control-3送转录组测序, 分别获得74 582 409和75 754 693个clean reads (表 3)。差异表达基因共有4 081个, 其中1 738个基因表达上调, 2 343个基因表达下调(图 7A)。KEGG富集分析表明差异表达基因显著富集在次生代谢物的生物合成(ko01110)、苯丙烷类生物合成(ko00940)、代谢途径(ko01100) 和植物激素信号转导(ko04075) 等(图 7B)。丹参毛状根中过表达SmHPPR1后, 引起了丹酚酸生物合成途径基因表达量变化, 如SmTAT2表达量上调; Sm4CL4、Sm4CL6、Sm4CL9、SmRAS4-like、SmRAS6-like1和SmRAS6-like2表达量下调(图 1, 表 4)。

qPCR分析SmHPPR1-OE-3和control-3毛状根中SmHPPR1的表达水平, 结果表明SmHPPR1在SmHPPR1-OE-3中的表达水平是control-3的9.21倍(图 1)。

8 PPI分析

将SmHPPR1-OE-3和control-3的DEGs导入STRING数据库构建PPI网络(score > 0.80), 共包含55个节点和198条边, 其中CYT (TR74706_c0_g1)、NADP⁺ (TR26565_c0_g1) 和NADP⁺ (TR68771_c0_g1) 占据网络中心位置, degree值分别是13、11和11 (图 8)。CYT (TR74706_c0_g1) 注释为细胞色素b6-f复合铁硫亚基, NADP⁺ (TR26565_c0_g1) 和NADP⁺ (TR68771_c0_g1) 注释为甘油醛-3-磷酸脱氢酶(表 5)。进一步分析表明它们主要参与了代谢过程(Ko01100, Ko01120, Ko01200等)、遗传信息处理(Ko04141, Ko03040)、环境信息处理(Ko04010)、细胞过程(Ko04144) 等。

讨论

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前期本课题组从丹参中克隆了SmHPPR1, 系统进化树分析表明SmHPPR1与彩叶草CbHPPR亲缘关系最近, 其次是拟南芥AtHPPR2和AtHPPR3 (图 2)。在此基础上, 获得了携带SmHPPR1的转基因拟南芥植株, 在转基因拟南芥中的表达产物表明SmHPPR1可能参与了pHPL的生物合成反应(图 4D、E)。拟南芥的两个基因AtHPPR2和AtHPPR3编码具有明显HPPR活性的蛋白, 催化pHPP还原为pHPL^[13]。为进一步阐明SmHPPR1在丹参丹酚酸途径中的作用, 诱导了过表达SmHPPR1丹参毛状根, 选取SmHPPR1-OE-3和control-3毛状根继代培养并进行研究, 结果表明: 与control-3毛状根相比, 过表达SmHPPR1-OE-3毛状根中RA、SAB及总丹酚酸含量提高, 但差异不显著。彩叶草中过表达CbHPPR提高了RA含量, RNAi降低了RA含量^[25]; Ag⁺对RA、咖啡酸和阿魏酸的积累显著增加, 而SAB、DSU和肉桂酸的积累明显减少并推测SAB可能来源于DSU生物合成的分支途径^[26]。

转录组学分析显示过表达SmHPPR1丹参毛状根中不仅SmHPPR1的表达量上调, 酪氨酸途径中SmTAT2基因表达量也上调(图 1), 推测酪氨酸途径中SmHPPR1和SmTAT2能协同调控RA的生物合成, 提高丹参毛状根中丹酚酸的含量。在拟南芥中, HPPR2和HPPR3与TAT1一起构成了一个可能从酪氨酸到pHPL的保守生物合成途径, 可以在特定的植物群体中产生一些特定的代谢物, 如RA^[10]。夏枯草毛状根中反义表达PvTAT致使PvTAT表达水平降低, PvTAT酶活性降低, RA的积累显著减少^[27]。同时, 过表达SmHPPR1毛状根中SmRAS4-like、SmRAS6-like1和SmRAS6-like2表达量下调(图 1)。敲除丹参SmRAS降低丹参毛状根中RA和SAB的含量^[28]。RAS是BAHD酰基转移酶超家族的一员, 可以酰化多种底物, 如薰衣草的LaAT1具有广泛的底物耐受性, 可以催化酯和酰胺的形成^[29-31]。由于RAS催化底物的范围广泛, 推测SmRAS4-like、SmRAS6-like1和SmRAS6-like2可能不参与丹酚酸的生物合成。丹参毛状根中过表达Sm4CL2丹酚酸含量提高, SmRAS4-like和SmRAS6-like1表达量下调; 反义表达Sm4CL2丹酚酸含量降低, SmRAS4-like和SmRAS6-like2表达量下调^[32]。

SmHPPR1-OE-3毛状根中Sm4CL4、Sm4CL6、Sm4CL9表达量下调(图 1), 但木质素含量与control-3无显著差异(表 2), 推测Sm4CL4、Sm4CL6和Sm4CL9可能不参与木质素生物合成。过表达Ptc4CL1杨树中木质素含量与Ptc4CL1基因表达量和Ptc4CL1酶活性正相关^[33]; 反义表达Pt4CL1杨树中木质素含量下降, 并与Pt4CL1基因表达量相关^[34]; 拟南芥中4CL4有助于木质素的沉积^[35]; 水曲柳中过表达Fm4CL-like1提高了木质素含量^[36]。

丹参毛状根中过表达SmHPPR1如何影响丹酚酸途径其他基因的表达？据报道, 丹参植株中RNAi-PAL会影响C4H、4CL2和TAT的表达, 并降低RA和SAB的含量^[37]。将SmHPPR1-OE-3毛状根的差异表达基因构建PPI网络, 结果表明CYT (TR74706_c0_g1)、NADP⁺ (TR26565_c0_g1) 和NADP⁺ (TR68771_c0_g1) 是网络的中心节点并参与代谢过程、细胞过程等(图 8)。NADPH是酶催化反应中的重要辅助因子, 用来实现电子传递, 丹参SmHPPR包含一个假定的催化结构域和一个NAD(P)H结合基序, 定位于细胞质, 与胞质NADPH依赖的羟基丙酮酸还原酶(HPR) 在光呼吸过程中的作用类似, 可以在NADPH的存在下将pHPP还原为pHPL^[38], 这一结果与本课题组的蛋白互作结果相类似。认为植物次生代谢产物的不同生物合成途径之间可能存在一个复杂的蛋白-蛋白相互作用网络, SmHPPR1-OE-3毛状根中其他通路基因的协同转录调控可能是来自合成途径基因相应酶的蛋白-蛋白相互作用的结果。

Xiao等^[39]报道丹参毛状根中过表达单个C4H, TAT和HPPR; 共表达TAT/HPPR和抑制表达4-羟基苯基丙酮酸双加氧酶(HPPD) 均能提高RA和丹酚酸B含量。本文在丹参毛状根中过表达单个SmHPPR1, 其序列与Xiao等^[39]报道的HPPR (GenBank: DQ099741) 序列98.4%同源。经过长达10年的跟踪研究, 结果表明过表达SmHPPR1能提高RA和丹酚酸B含量但差异不显著, 且毛状根性状能较稳定遗传, 与Xiao等^[39]报道的结果一致。进一步从转录组水平上分析发现: 丹参毛状根中过表达SmHPPR1引起了丹酚酸含量提高可能与协同调控了其他苯丙烷类途径基因如SmTAT2的表达水平有关。同时, 本实验室在丹参毛状根中过表达和反义表达Sm4CL2, 结果表明过表达毛状根(Sm4CL2-OE-4) 的丹酚酸含量是对照毛状根(control-3) 的1.35倍, 反义表达毛状根(anti-Sm4CL2-1) 的丹酚酸含量是对照毛状根(control-3) 的37.32%^[32], 而Sm4CL2和SmHPPR1分别是苯丙氨酸和酪氨酸分支途径中的最后一个关键酶, 推测与Sm4CL2相比, 丹参毛状根中SmHPPR1对丹酚酸含量贡献相对较小。

综上所述, SmHPPR1在携带SmHPPR1转基因的拟南芥中参与了4-羟基苯乳酸的生物合成反应, 丹参毛状根中过表达SmHPPR1引起了其他苯丙烷类途径基因表达水平的改变, 从而增加了丹酚酸含量。生物合成途径基因相对应的酶之间的蛋白-蛋白相互作用可能参与了部分代谢通路基因之间的协同转录调控。

致谢: 本论文转基因拟南芥工作得到中国科学院分子植物科学卓越创新中心/植物生理生态研究所何玉科研究员的支持, 在此表示感谢。

作者贡献: 赵淑娟设计了本实验及论文的修改; 谈荣慧诱导了丹参毛状根、qPCR实验、木质素含量测定、数据分析和论文初稿的写作; 赵望构建了植物表达载体、诱导转SmHPPR1拟南芥及其酚酸类物质含量测定; 张金家完成了丹参毛状根中丹酚酸的含量测定。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然科学青年科学基金资助项目(30300447)
上海中医药大学预算内项目(2019LK103)

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2023年第58卷第9期

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doi: 10.16438/j.0513-4870.2023-0030

接收时间：2023-01-10
首发时间：2025-11-21
出版时间：2023-09-12

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收稿日期：2023-01-10
修回日期：2023-02-24

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国家自然科学青年科学基金资助项目(30300447)

上海中医药大学预算内项目(2019LK103)

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*赵淑娟, Tel: 86-21-51322576, E-mail: zhaoshujuan@126.com; zhaoshujuan@shutcm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Primer name	Primer sequence (5′-3′)	Fragment length /bp
Screening primers of overexpression hairy roots
SmHPPR1-F1	ttggatccaccactcaaacaccatct	1 019
SmHPPR1-R1	tgaagctttcaaagagttatcaaac
SmHPPR1-F2	tcaggatccatggaggcgatcggtgttc	933
SmHPPR1-R2	cagtctagaaggtgttaacagaggct
Kan-F	cacaacagacaatcggc	660
Kan-R	cgtaaagcacgaggaag
qPCR primes
qActin-F	aggaaccaccgatccagaca	278
qActin-R	ggtgccctgaggtcctgtt
qSmHPPR1-F	cgccgacgccgatatcatcgactca	190
qSmHPPR1-R	aatccgcctcagaaccgccagcat

Primer name

Primer sequence (5′-3′)

Fragment length /bp

Screening primers of overexpression hairy roots

SmHPPR1-F1

ttggatccaccactcaaacaccatct

1 019

SmHPPR1-R1

tgaagctttcaaagagttatcaaac

SmHPPR1-F2

tcaggatccatggaggcgatcggtgttc

933

SmHPPR1-R2

cagtctagaaggtgttaacagaggct

Kan-F

cacaacagacaatcggc

660

Kan-R

cgtaaagcacgaggaag

qPCR primes

qActin-F

aggaaccaccgatccagaca

278

qActin-R

ggtgccctgaggtcctgtt

qSmHPPR1-F

cgccgacgccgatatcatcgactca

190

qSmHPPR1-R

aatccgcctcagaaccgccagcat

Sample	Sampling time	Dry weight/g	Salvianolic acid/mg·g^-1 DW	Lignin/%
Control-3	2010	0.56	/	45.45	34.51	79.96	/
2022	0.96 ± 0.11	1.71 ± 0.37	67.42 ± 3.70	23.46 ± 4.20	90.88 ± 7.45	11.38 ± 0.37
SmHPPR1-OE-3	2008	0.61	/	60.28	22.64	82.92	/
2022	0.73 ± 0.01	0.62 ± 0.07	73.65 ± 4.85	30.51 ± 2.78	104.16 ± 4.28	12.70 ± 1.36

Sample

Sampling time

Dry weight/g

Salvianolic acid/mg·g^-1 DW

Lignin/%

DSU

SAB

TSA

Control-3

2010

0.56

45.45

34.51

79.96

2022

0.96 ± 0.11

1.71 ± 0.37

67.42 ± 3.70

23.46 ± 4.20

90.88 ± 7.45

11.38 ± 0.37

SmHPPR1-OE-3

2008

0.61

60.28

22.64

82.92

2022

0.73 ± 0.01

0.62 ± 0.07

73.65 ± 4.85

30.51 ± 2.78

104.16 ± 4.28

12.70 ± 1.36

Sample	Raw_read	Clean_read	Error/%	Q20/%	Q30/%	GC/%
Control-3-①	82 142 460	80 974 024	0.02	98.18	94.45	48.10
Control-3-②	71 304 730	70 535 362	0.02	98.20	94.50	48.22
SmHPPR1-OE-3-①	75 248 990	74 743 574	0.02	98.24	94.58	47.39
SmHPPR1-OE-3-②	74 884 956	74 421 244	0.02	98.43	95.1	47.37

Sample

Raw_read

Clean_read

Error/%

Q20/%

Q30/%

GC/%

Control-3-①

82 142 460

80 974 024

0.02

98.18

94.45

48.10

Control-3-②

71 304 730

70 535 362

0.02

98.20

94.50

48.22

SmHPPR1-OE-3-①

75 248 990

74 743 574

0.02

98.24

94.58

47.39

SmHPPR1-OE-3-②

74 884 956

74 421 244

0.02

98.43

95.1

47.37

Gene	Gene_id	log₂FC	NR_annotation
SmTAT2	TR739_c4_g1	1.05	Tyrosine aminotransferase 2 [Salvia miltiorrhiza]
Sm4CL4	TR174_c0_g1	-1.28	4-Coumarate: coenzyme A ligase 4 [Salvia miltiorrhiza]
Sm4CL6	TR4603_c0_g1	-2.17	4-Coumarate: coenzyme A ligase 6, partial [Salvia miltiorrhiza]
SmRAS4-like	TR5466_c0_g1	-3.00	Rosmarinic acid synthase 4, partial [Salvia miltiorrhiza]
Sm4CL9	TR8805_c0_g1	-3.53	4-Coumarate--CoA ligase 9[Salvia miltiorrhiza]
SmRAS6-like1	TR2028_c4_g1	-4.08	Rosmarinic acid synthase 6 [Salvia miltiorrhiza]
SmRAS6-like2	TR35281_c0_g1	-6.49	Rosmarinic acid synthase 6 [Salvia miltiorrhiza]

Gene

Gene_id

log₂FC

NR_annotation

SmTAT2

TR739_c4_g1

1.05

Tyrosine aminotransferase 2 [Salvia miltiorrhiza]

Sm4CL4

TR174_c0_g1

-1.28

4-Coumarate: coenzyme A ligase 4 [Salvia miltiorrhiza]

Sm4CL6

TR4603_c0_g1

-2.17

4-Coumarate: coenzyme A ligase 6, partial [Salvia miltiorrhiza]

SmRAS4-like

TR5466_c0_g1

-3.00

Rosmarinic acid synthase 4, partial [Salvia miltiorrhiza]

Sm4CL9

TR8805_c0_g1

-3.53

4-Coumarate--CoA ligase 9[Salvia miltiorrhiza]

SmRAS6-like1

TR2028_c4_g1

-4.08

Rosmarinic acid synthase 6 [Salvia miltiorrhiza]

SmRAS6-like2

TR35281_c0_g1

-6.49

Rosmarinic acid synthase 6 [Salvia miltiorrhiza]

Gene	Gene_id	log₂FC	NR_annotation
CYT	TR74706_c0_g1	-1.59	Cytochrome b6-f complex iron-sulfur subunit [Salvia splendens]
NADP+	TR26565_c0_g1	-1.92	Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) [Salvia splendens]
NADP+	TR68771_c0_g1	-2.91	Glyceraldehyde-3-phosphate dehydrogenase (NADP+) [Salvia splendens]

Gene

Gene_id

log₂FC

NR_annotation

CYT

TR74706_c0_g1

-1.59

Cytochrome b6-f complex iron-sulfur subunit [Salvia splendens]

NADP+

TR26565_c0_g1

-1.92

Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) [Salvia splendens]

NADP+

TR68771_c0_g1

-2.91

Glyceraldehyde-3-phosphate dehydrogenase (NADP+) [Salvia splendens]