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，国家生化工程技术研究中心（上海），200237

基金项目: 国家重大专项项目（No.2020YFA0906800）资助项目

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：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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- 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

Optimization of Callus Suspension Culture Medium for enhancing Resveratrol Biosynthesis in Grape (Vitis vinifera)

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

摘要

摘要: 建立和优化了一种醉金香葡萄悬浮细胞制备白藜芦醇的最优化营养配置工艺。以细胞生长和白藜芦醇生物合成量为响应值，通过单因素试验、Plackett-Burman、响应面试验详细考察了不同碳源、氮源、前体、诱导剂和激素等因素对醉金香葡萄细胞白藜芦醇合成的影响。研究结果获得了醉金香葡萄悬浮细胞的最佳培养条件，在此条件下，葡萄细胞悬浮培养得到单位细胞白藜芦醇最高表达量为3026.64±56 μg/g，与对照相比提高了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 L. 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 800 mg/L, phenylalanine 100 mg/L, methyl jasmonate 80 μmol/L, 2, 4-chlorophenoxyacetic acid 4.1 mg/L, 6-benzylaminopurine 0.2 mg/L, polyvinylpyrrolidone 1g/L. Under this condition, the highest expression of resveratrol was 3026.64 ± 56 μg/g in cell suspension culture at 26 °C for 7 days, 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 detail, 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 enlarging the production of resveratrol and other substances.
- Vitis vinifera /
- Optimization of 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

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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. The effects of different precursors and inducers on cell growth and production of Vitis vinifera

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图 4 MeJA添加时间对醉金香葡萄细胞生长及产物合成的影响

Figure 4. the 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. Different hormone types and combinations on cell growth and production of Vitis vinifera L

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图 6 P-B各实验组中残糖浓度和醉金香葡萄细胞生物量情况

Figure 6. Residual sugar concentration and cell biomass of Vitis vinifera L. in P-B experimental groups

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图 7 酵母提取物、茉莉酸甲酯和2,4-D含量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 contents on resveratrol synthesis

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表 1 Plackett-Burman培养组分和水平表头设计

Table 1. The culture components and levels in Plackett-Burman design

Factor	Code	level
Factor	Code	low(−1)	high(1)
B5 (g/L)	X1	1	5
Sucrose (g/L)	X2	24	37.5
PVP (g/L)	X3	1	3
Yeast extract (mg/L)	X4	100	1000
Phe (mg/L)	X5	10	100
MeJA (μmol/L)	X6	10	100
6-BA (mg/L)	X7	0.2	1
2,4-D (mg/L)	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. The factors and levels in central-composite design

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

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表 3 Plachett-Burman设计实验结果

Table 3. The experiment results in Plackett–Burman design

Run	Variable level											Res(μg/g)
Run	X1	X2	X3	X4	X5	X6	X7	X8	X9	X10	X11	Res(μg/g)
1	1	−1	1	1	1	−1	−1	−1	1	−1	1	1550
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
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
10	−1	1	1	−1	1	1	1	−1	−1	−1	1	1850
11	1	1	−1	1	1	1	−1	−1	−1	1	−1	1875
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. The regression results for the Plackett-Burman design

Factor	Sum of Squares	df	Mean square	F value	Prob > F	Significantce	Importance ranking
Model	4.37×10⁶	9	4.86×10⁵	2056.56	0.0005	Significant
E-MeJA	3.52×10⁶	1	3.52×10⁶	14903.65	<0.0001		1
F-2,4-D	2.78×10⁵	1	2.78×10⁵	1175.57	0.0008		2
C-Yeast extract	2.39×10⁵	1	2.39×10⁵	1011.37	0.001		3
H-PVP	1.77×10⁵	1	34550.4	750.96	0.0013		4
G-6-BA	80508.52	1	80508.52	340.73	0.029		5

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表 5 最陡爬坡试验设计及结果

Table 5. design and results in steepest ascent path

Group	ρ(Yeast extract)/ (mg·L⁻¹)	C(MeJA)/ (μmol·L⁻¹)	ρ(2,4-D)/ (mg·L⁻¹)	Res/ (μg·g⁻¹)
1	550	55	3	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	4.1	3100
7	850	85	4.32	2940
8	900	90	4.54	2900
9	950	95	4.76	2790
10	1	100	5	2730

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表 6 CCD响应面设计及结果

Table 6. The design and results of the central composite experiment

Run	ρ(Yeast extract)/ (mg·L⁻¹)	C(MeJA)/ (μmol·L⁻¹)	ρ(2,4-D)/ (mg·L⁻¹)	Res/ (μg·g⁻¹)
Run	A	B	C	Y
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
7	700	90	4.6	2010.4
8	900	90	4.6	2254.6
9	631.82	80	4.1	2550.02
10	968.18	80	4.1	2650
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	3271.02
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	3299.42
20	800	80	4.1	3280.4

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表 7 CCD试验方差分析

Table 7. The variance analysis of central-composite design

Source	Sum of squares	Mean square	F Value	Prob > F
Model	5.15×10⁶	5.74×10⁵	17.45	<0.0001
A-Yeast extract	9.23×10³	9.23×10³	0.28	0.6074
B-MeJA	1.06×10⁵	1.06×10⁵	3.22	0.1028
C-2,4-D	314.72	314.72	9.59×10⁻³	0.9239
AB	3.58×10⁵	3.58×10⁵	10.9	0.008
AC	8051.8	8051.8	0.25	0.631
BC	1220.18	1220.18	0.037	0.8509
A2	9.65×10⁵	9.65×10⁵	29.4	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	3545.11	709.02
Cor Total	5.48×10⁶
R²= 0.9401; Adj R²=0.8862

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