生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 75-84.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0143
张秀民1(), 马绍英2, 杨洁1, 包金玉1, 张晓玲1, 田鹏1, 路亚琦1, 李胜1()
收稿日期:
2021-02-03
出版日期:
2021-08-26
发布日期:
2021-09-10
作者简介:
张秀民,女,硕士研究生,研究方向:植物学;E-mail: 基金资助:
ZHANG Xiu-min1(), MA Shao-ying2, YANG Jie1, BAO Jin-yu1, ZHANG Xiao-ling1, TIAN Peng1, LU Ya-qi1, LI Sheng1()
Received:
2021-02-03
Published:
2021-08-26
Online:
2021-09-10
摘要:
本研究旨在探究不同单因素条件下西兰花毛状根培养体系中萝卜硫苷(glucoraphanin,GRA)和萝卜硫素(sulforaphane,SF)释放的关系。通过10 mmol/L茉莉酸甲酯(methyl jasmonate,MeJA)处理,探究了在不同接种量、培养基体积、pH、培养温度及摇床转速下对西兰花毛状根中GRA和SF释放的影响;在单因素实验基础上,选取培养温度、毛状根接种量及pH进行响应面实验。结果表明:当培养温度为25.44℃,毛状根接种量为0.15 g,pH 5.56,西兰花毛状根中GRA和SF总产量为2.08 mg/flask,与回归模型预测理论总产量2.44 mg/flask相近;不同培养条件下,西兰花毛状根培养体系中GRA和SF释放至培养基中的量随之不同。
张秀民, 马绍英, 杨洁, 包金玉, 张晓玲, 田鹏, 路亚琦, 李胜. 以次生代谢物产量为目标的西兰花毛状根培养技术体系优化[J]. 生物技术通报, 2021, 37(8): 75-84.
ZHANG Xiu-min, MA Shao-ying, YANG Jie, BAO Jin-yu, ZHANG Xiao-ling, TIAN Peng, LU Ya-qi, LI Sheng. Optimization of Hairy Roots Culture System of Broccoli Aiming at the Yield of Secondary Metabolites[J]. Biotechnology Bulletin, 2021, 37(8): 75-84.
因素 Factor | 因素Factor | |||||
---|---|---|---|---|---|---|
接种量 Inoculation amount/g | 培养基体积 Medium volume/mL | 转速 Rotating speed/(r·min-1) | 温度 Temperature/℃ | pH | ||
接种量Inoculation amount/g | 0.05 0.1 0.15 0.2 0.25 | 100 | 110 | 25 | 6.0 | |
培养基体积Medium volume/mL | 0.15 | 80 90 100 110 120 | 110 | 25 | 6.0 | |
转速Rotating speed/(r·min-1) | 0.15 | 100 | 90 100 110 120 130 | 25 | 6.0 | |
温度Temperature/℃ | 0.15 | 100 | 110 | 21 23 25 27 29 | 6.0 | |
pH | 0.15 | 100 | 110 | 25 | 5.0 5.5 6.0 6.5 7.0 |
表1 单因素实验设计
Table 1 Single factor experimental design
因素 Factor | 因素Factor | |||||
---|---|---|---|---|---|---|
接种量 Inoculation amount/g | 培养基体积 Medium volume/mL | 转速 Rotating speed/(r·min-1) | 温度 Temperature/℃ | pH | ||
接种量Inoculation amount/g | 0.05 0.1 0.15 0.2 0.25 | 100 | 110 | 25 | 6.0 | |
培养基体积Medium volume/mL | 0.15 | 80 90 100 110 120 | 110 | 25 | 6.0 | |
转速Rotating speed/(r·min-1) | 0.15 | 100 | 90 100 110 120 130 | 25 | 6.0 | |
温度Temperature/℃ | 0.15 | 100 | 110 | 21 23 25 27 29 | 6.0 | |
pH | 0.15 | 100 | 110 | 25 | 5.0 5.5 6.0 6.5 7.0 |
因素Factor | -1 | 0 | 1 |
---|---|---|---|
A:培养温度Culture temperature/℃ | 23 | 25 | 27 |
B:接种量Inoculation amount/g | 0.1 | 0.15 | 0.2 |
C:pH | 5 | 5.5 | 6 |
表2 Box-behnken试验因素水平表
Table 2 Box-behnken test factor levels
因素Factor | -1 | 0 | 1 |
---|---|---|---|
A:培养温度Culture temperature/℃ | 23 | 25 | 27 |
B:接种量Inoculation amount/g | 0.1 | 0.15 | 0.2 |
C:pH | 5 | 5.5 | 6 |
图1 接种量对西兰花毛状根培养体系中GRA和SF释放的影响 A:GRA产量;B:SF产量;C:MYR活性,图中不同小写字母表示差异显著(P≤0.05),下同
Fig. 1 Effects of inoculation amount on the GRA and SF release in broccoli hairy root culture system A:GRA yield. B:SF yield. C:MYR activity,different small letters in the figure indicate significant difference(P ≤0.05),the same below
序号 Sequence number | A:接种量 Inoculation amount/g | B:培养温度 Culture temperature/℃ | C:pH | GRA及SF总产量 Total yield of GRA and SF /mg |
---|---|---|---|---|
1 | 0.10 | 23 | 5.5 | 1.18 |
2 | 0.10 | 27 | 5.5 | 1.61 |
3 | 0.20 | 23 | 5.5 | 1.80 |
4 | 0.20 | 27 | 5.5 | 1.71 |
5 | 0.15 | 23 | 5 | 1.51 |
6 | 0.15 | 27 | 5 | 1.31 |
7 | 0.15 | 23 | 6 | 1.71 |
8 | 0.15 | 27 | 6 | 1.81 |
9 | 0.10 | 25 | 5 | 1.75 |
10 | 0.20 | 25 | 5 | 1.76 |
11 | 0.10 | 25 | 6 | 1.40 |
12 | 0.20 | 25 | 6 | 1.88 |
13 | 0.15 | 25 | 5.5 | 2.61 |
14 | 0.15 | 25 | 5.5 | 2.48 |
15 | 0.15 | 25 | 5.5 | 2.18 |
16 | 0.15 | 25 | 5.5 | 2.31 |
17 | 0.15 | 25 | 5.5 | 2.58 |
表3 响应面设计与实验结果
Table 3 Response surface design and experimental results
序号 Sequence number | A:接种量 Inoculation amount/g | B:培养温度 Culture temperature/℃ | C:pH | GRA及SF总产量 Total yield of GRA and SF /mg |
---|---|---|---|---|
1 | 0.10 | 23 | 5.5 | 1.18 |
2 | 0.10 | 27 | 5.5 | 1.61 |
3 | 0.20 | 23 | 5.5 | 1.80 |
4 | 0.20 | 27 | 5.5 | 1.71 |
5 | 0.15 | 23 | 5 | 1.51 |
6 | 0.15 | 27 | 5 | 1.31 |
7 | 0.15 | 23 | 6 | 1.71 |
8 | 0.15 | 27 | 6 | 1.81 |
9 | 0.10 | 25 | 5 | 1.75 |
10 | 0.20 | 25 | 5 | 1.76 |
11 | 0.10 | 25 | 6 | 1.40 |
12 | 0.20 | 25 | 6 | 1.88 |
13 | 0.15 | 25 | 5.5 | 2.61 |
14 | 0.15 | 25 | 5.5 | 2.48 |
15 | 0.15 | 25 | 5.5 | 2.18 |
16 | 0.15 | 25 | 5.5 | 2.31 |
17 | 0.15 | 25 | 5.5 | 2.58 |
方差来源Source of variance | 平方和Sum of squares | 自由度df | 均方Means square | F | P | 显著性Significance |
---|---|---|---|---|---|---|
模型Model | 2.73 | 9 | 0.30 | 7.78 | 0.0065 | ** |
A-培养温度Culture temperature | 0.00072 | 1 | 0.00072 | 0.18 | 0.6805 | |
B-接种量Inoculation amount | 0.18 | 1 | 0.18 | 4.69 | 0.0671 | |
C-pH | 0.028 | 1 | 0.028 | 0.71 | 0.4281 | |
AB | 0.068 | 1 | 0.068 | 1.73 | 0.2296 | |
AC | 0.022 | 1 | 0.022 | 0.58 | 0.4725 | |
BC | 0.055 | 1 | 0.055 | 1.41 | 0.2730 | |
A2 | 0.99 | 1 | 0.99 | 25.35 | 0.0015 | ** |
B2 | 0.58 | 1 | 0.58 | 14.95 | 0.0062 | ** |
C2 | 0.55 | 1 | 0.55 | 14.16 | 0.0071 | ** |
残差Residual | 0.27 | 7 | 0.039 | |||
失拟项Lack of fit | 0.14 | 3 | 0.046 | 1.38 | 0.3701 | |
纯误差Pure error | 0.13 | 4 | 0.034 | |||
总值Total value | 3.01 | 16 | ||||
R2=0.9091 R2Adj=0.8629 |
表4 响应面方差分析
Table 4 Analysis of variance response surface
方差来源Source of variance | 平方和Sum of squares | 自由度df | 均方Means square | F | P | 显著性Significance |
---|---|---|---|---|---|---|
模型Model | 2.73 | 9 | 0.30 | 7.78 | 0.0065 | ** |
A-培养温度Culture temperature | 0.00072 | 1 | 0.00072 | 0.18 | 0.6805 | |
B-接种量Inoculation amount | 0.18 | 1 | 0.18 | 4.69 | 0.0671 | |
C-pH | 0.028 | 1 | 0.028 | 0.71 | 0.4281 | |
AB | 0.068 | 1 | 0.068 | 1.73 | 0.2296 | |
AC | 0.022 | 1 | 0.022 | 0.58 | 0.4725 | |
BC | 0.055 | 1 | 0.055 | 1.41 | 0.2730 | |
A2 | 0.99 | 1 | 0.99 | 25.35 | 0.0015 | ** |
B2 | 0.58 | 1 | 0.58 | 14.95 | 0.0062 | ** |
C2 | 0.55 | 1 | 0.55 | 14.16 | 0.0071 | ** |
残差Residual | 0.27 | 7 | 0.039 | |||
失拟项Lack of fit | 0.14 | 3 | 0.046 | 1.38 | 0.3701 | |
纯误差Pure error | 0.13 | 4 | 0.034 | |||
总值Total value | 3.01 | 16 | ||||
R2=0.9091 R2Adj=0.8629 |
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