Optimization of Callus Suspension Culture Medium for Enhancing Resveratrol Biosynthesis in Vitis vinifera Grape
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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。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.
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Key words:
- Vitis vinifera /
- callus cell /
- suspension culture /
- nutrient components /
- resveratrol
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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 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 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) 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 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)X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 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 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 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 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 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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