醉金香葡萄愈伤细胞悬浮培养基优化促进白藜芦醇的合成

王晓惠; 王泽建; 肖慈英; 刘泽波; 郭美锦; 庄英萍

doi:10.14135/j.cnki.1006-3080.20210221002

醉金香葡萄愈伤细胞悬浮培养基优化促进白藜芦醇的合成

doi: 10.14135/j.cnki.1006-3080.20210221002

华东理工大学生物反应器工程国家重点实验室，上海 200237

基金项目: 国家重点研发专项（2020YFA0906800）

详细信息

作者简介:
王晓惠（1996—），女，山东人，硕士生，主要研究方向为植物细胞培养。E-mail：wxhui1010@163.com

通讯作者:
庄英萍，E-mail：ypzhuang@ecust.edu.cn

中图分类号: TS201.3
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- 文章访问数: 231
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- 被引次数: 0
出版历程
- 收稿日期: 2021-02-21
- 网络出版日期: 2021-06-29
- 刊出日期: 2022-04-22

Optimization of Callus Suspension Culture Medium for Enhancing Resveratrol Biosynthesis in Vitis vinifera Grape

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

摘要

摘要: 建立和优化了一种醉金香葡萄悬浮细胞合成白藜芦醇的工艺。以细胞生长和白藜芦醇生物合成量为响应值，通过单因素实验、Plackett-Burman、响应面实验考察了不同碳源、氮源、前体、诱导剂和激素等因素对醉金香葡萄细胞悬浮培养基合成白藜芦醇的影响。结果表明，在醉金香葡萄悬浮细胞的最佳培养条件下，单位葡萄细胞中白藜芦醇的最高表达量为（3026.64±56）μg/g，与对照组相比提高了51.13倍，为后续实现利用醉金香葡萄细胞悬浮培养生产白藜芦醇提供了理论基础。
- 醉金香葡萄 /
- 愈伤细胞 /
- 悬浮培养 /
- 营养组分优化 /
- 白藜芦醇
Abstract: Resveratrol, a secondary metabolite of stilbene, is synthesized by the L-Phenylalanine pathway in plants. Under external conditions, various plants are used to produce resveratrol. However, the most commonly used plant cell cultures for the production of stilbene are still from grapes. In this paper, an optimal technology for the production of resveratrol from suspension cell of Vitis vinifera Loes. was established. In response to cell growth and resveratrol biosynthesis, the effects of different carbon sources, nitrogen sources, precursors, inducers and hormones on resveratrol synthesis in the grape cells were studied by single factor test, Plackett-Burman test and response surface methodology. The results showed that the optimal nutrient conditions of suspension cells were as follows: B5 medium 5 g/L, sucrose 37.5 g/L, yeast extract 900 mg/L, phenylalanine 100 mg/L, methyl jasmonate 84 μmol/L, 2, 4-chlorophenoxyacetic acid 4.1 mg/L, 6-benzylaminopurine 0.2 mg/L, polyvinylpyrrolidone 1.0 g/L. Under these conditions, the highest expression of resveratrol was (3026.64 ± 56)μg/g in cell suspension culture at 26 °C for 7 d, which was 51.13 times higher than that before optimization, the optimal nutrient conditions can significantly promote the synthesis of resveratrol. In this study, the nutrient composition of suspension cells of Vitis vinifera was studied in details, and the optimal culture conditions for the growth and resveratrol synthesis of the suspension cells were determined. On the one hand, it provides a basis for analyzing the function of grape suspension cells, and on the other hand, it lays a good foundation for scale-up the production of resveratrol and other substances.
- Vitis vinifera /
- callus cell /
- suspension culture /
- nutrient components /
- resveratrol

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图 1 不同碳源底物对醉金香葡萄细胞生长及产物合成的影响

Figure 1. Effects of substrates with different carbon sources on cell growth and production of Vitis vinifera

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图 2 不同氮源底物对醉金香葡萄细胞生长及产物合成的影响

Figure 2. Effects of substrates with different nitrogen sources on cell growth and production of Vitis vinifera

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图 3 不同前体和诱导剂对醉金香葡萄细胞生长及产物合成的影响

Figure 3. Effects of different precursors and inducers on cell growth and production of Vitis vinifera

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图 4 茉莉酸甲酯添加时间对醉金香葡萄细胞生长及产物合成的影响

Figure 4. Effect of adding time of MeJA on the cell growth and product synthesis of Vitis vinifera

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图 5 不同激素类型和激素组合对醉金香葡萄细胞生长及产物合成的影响

Figure 5. Effect of different hormone types and combinations on cell growth and production of Vitis vinifera

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图 6 Plackett-Burman各实验组中白藜芦醇含量和醉金香葡萄细胞生长情况

Figure 6. Resveratrol biomass and cell of Vitis vinifera in Plackett-Burman experimental groups

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图 7 酵母提取物、茉莉酸甲酯和2,4-D交互作用对白藜芦醇合成影响的响应面和等高线

Figure 7. Response surface and contour of the interaction of yeast extract, methyl jasmonate and 2,4-D on resveratrol synthesis

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表 1 Plackett-Burman培养组分和水平

Table 1. Culture components and levels in Plackett-Burman

Factor	Code	Level
Factor	Code	Low(−1)	High(1)
B5	X₁	1	5
Sucrose	X₂	24	37.5
PVP	X₃	1	3
Yeast extract	X₄	100	1000
Phe	X₅	10	100
MeJA	X₆	10	100
6-BA	X₇	0.2	1
2,4-D	X₈	1	5
Blank term	X₉	−1	1
Blank term	X₁₀	−1	1
Blank term	X₁₁	−1	1

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表 2 Central-Composite设计的因素水平

Table 2. Factors and levels of central-composite design

Code value	$\rho $/（mg·L⁻¹）
Code value	Yeast extract	MeJA	2,4-D
−1.68	631.82	63.18	3.26
−1	700	70	3.6
0	800	80	4.1
1	900	90	4.6
1.68	968.18	96.82	4.94

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表 3 Plachett-Burman设计试验结果

Table 3. Experiment results in Plackett–Burman design

Group	Variable level											Resveratrol biomass/(μg·g⁻¹)
Group	X₁	X₂	X₃	X₄	X₅	X₆	X₇	X₈	X₉	X₁₀	X₁₁	Resveratrol biomass/(μg·g⁻¹)
1	1	−1	1	1	1	−1	−1	−1	1	−1	1	1550.00
2	1	−1	−1	−1	1	−1	1	1	−1	1	1	1001.85
3	−1	1	1	1	−1	−1	−1	1	−1	1	1	460.65
4	−1	−1	1	−1	1	1	−1	1	1	1	−1	1780.00
5	1	−1	1	1	−1	1	1	1	−1	−1	−1	467.95
6	−1	1	−1	1	1	−1	1	1	1	−1	1	1035.35
7	−1	−1	−1	−1	−1	−1	−1	−1	1	1	−1	301.20
8	1	1	−1	−1	−1	1	−1	1	−1	−1	1	366.50
9	1	1	1	−1	−1	−1	1	−1	1	1	−1	580.00
10	−1	1	1	−1	1	1	1	−1	−1	−1	1	1850.00
11	1	1	−1	1	1	1	−1	−1	−1	1	−1	1875.00
12	−1	−1	−1	1	−1	1	1	−1	1	1	1	415.30
13	0	0	0	0	0	0	0	0	0	0	0	915.35
X₁—B5；X₂—Sucrose；X₃—Yeast extract；X₄—Phe；X₅—MeJA; X₆—2,4-D；X₇—6-BA；X₈—PVP；X₉~X₁₁ —Error

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表 4 Plachett-Burman设计各因数效应分析

Table 4. Regression results for the Plackett-Burman design

Factor	Sum of squares	Degree of freedom	Mean square	F value	P	Importance ranking
Model	4.37×10⁶	9	4.86×10⁵	2056.56	0.0005	—
MeJA	3.52×10⁶	1	3.52×10⁶	14903.65	<0.0001	1
2,4-D	2.78×10⁵	1	2.78×10⁵	1175.57	0.0008	2
Yeast extract	2.39×10⁵	1	2.39×10⁵	1011.37	0.001	3
PVP	1.77×10⁵	1	3.46×10⁴	750.96	0.0013	4
6-BA	8.05×10⁴	1	8.05×10⁴	340.73	0.029	5

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表 5 最陡爬坡实验设计及结果

Table 5. Design and result in steepest ascent path

Group	ρ(Yeast extract)/ (mg·L⁻¹)	c(MeJA)/ (μmol·L⁻¹)	ρ(2,4-D)/ (mg·L⁻¹)	Resveratrol biomass/(μg·g⁻¹)
1	550	55	3.00	1690
2	600	60	3.22	1740
3	650	65	3.44	1860
4	700	70	3.66	2310
5	750	75	3.88	2760
6	800	80	4.10	3100
7	850	85	4.32	2940
8	900	90	4.54	2900
9	950	95	4.76	2790
10	1000	100	5.00	2730

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表 6 CCD响应面设计及结果

Table 6. Design and result of the central composite experiment

Group	ρ(Yeast extract)/ (mg·L⁻¹)	c(MeJA)/ (μmol·L⁻¹)	ρ(2,4-D)/ (mg·L⁻¹)	Resveratrol biomass/(μg·g⁻¹)
1	700	70	3.6	2460.2
2	900	70	3.6	1985.6
3	700	90	3.6	2020.5
4	900	90	3.6	2360.4
5	700	70	4.6	2530.7
6	900	70	4.6	1898.0
7	700	90	4.6	2010.4
8	900	90	4.6	2254.6
9	631.82	80	4.1	2550.0
10	968.18	80	4.1	2650.0
11	800	63.18	4.1	1899.5
12	800	96.82	4.1	2750.1
13	800	80	3.26	2080.4
14	800	80	4.94	2120.5
15	800	80	4.1	3271.0
16	800	80	4.1	3250.7
17	800	80	4.1	3240.2
18	800	80	4.1	3308.2
19	800	80	4.1	3299.4
20	800	80	4.1	3280.4

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表 7 CCD试验方差分析

Table 7. Variance analysis of central-composite design

Source	Sum of squares	Mean square	F	P
Model	5.15×10⁶	5.74×10⁵	17.45	<0.0001
Yeast extract	9.23×10³	9.23×10³	0.28	0.6074
MeJA	1.06×10⁵	1.06×10⁵	3.22	0.1028
2,4-D	314.72	314.72	9.59×10⁻³	0.9239
AB	3.58×10⁵	3.58×10⁵	10.90	0.0080
AC	8051.80	8051.80	0.25	0.6310
BC	1220.18	1220.18	0.037	0.8509
A2	9.65×10⁵	9.65×10⁵	29.40	0.0003
B2	1.83×10⁶	1.83×10⁶	55.67	<0.0001
C2	2.73×10⁶	2.73×10⁶	83.24	<0.0001
Residual	3.28×10⁵	32810.75
Lack of fit	3.25×10⁵	6.49×10⁴	91.55	<0.0001
Pure error	355.11	709.02
Cor total	5.48×10⁶
R²= 0.9401; Adj R²=0.8862

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