植物学报 ›› 2022, Vol. 57 ›› Issue (3): 340-349.DOI: 10.11983/CBB21206
张玉琴1,2, 吴嘉诚1,2, 何萌2, 刘仁义3, 朱晓玥2,*()
收稿日期:
2021-11-24
接受日期:
2022-02-07
出版日期:
2022-05-01
发布日期:
2022-05-18
通讯作者:
朱晓玥
作者简介:
* E-mail: xiaoyuezhu@fafu.edu.cn基金资助:
Yuqin Zhang1,2, Jiacheng Wu1,2, Meng He2, Renyi Liu3, Xiaoyue Zhu2,*()
Received:
2021-11-24
Accepted:
2022-02-07
Online:
2022-05-01
Published:
2022-05-18
Contact:
Xiaoyue Zhu
摘要: 近年来, 茶树基因组测序的完成为茶树在分子和基因水平的研究奠定了基础。但由于转基因技术尚不成熟且茶树生长周期较长, 茶树的基因功能研究依然不能有效开展。采用铁观音(Camellia sinensis var. sinensis cv. ‘Tieguanyin’)实生幼苗叶片, 通过筛选多种纤维素酶、果胶酶、离析酶和甘露醇的浓度组合, 并结合原生质体的数量、活性和杂质含量综合确定了最佳配方, 成功建立了铁观音茶苗叶片原生质体提取和PEG介导的高效瞬时转化体系, 转化率达56.25%。利用该系统探索了茶氨酸代谢通路中2个重要合成酶(茶氨酸合成酶(TSI)和谷氨酰胺合成酶(GSII-1.1))的亚细胞定位。研究发现, 这2种酶均定位于铁观音原生质体细胞质中。茶苗叶片原生质体提取和瞬时转化体系的建立为茶树基因组功能研究奠定了技术基础。
张玉琴, 吴嘉诚, 何萌, 刘仁义, 朱晓玥. 铁观音原生质体高效瞬时转化方法的建立. 植物学报, 2022, 57(3): 340-349.
Yuqin Zhang, Jiacheng Wu, Meng He, Renyi Liu, Xiaoyue Zhu. An Efficient Protoplast Transient Expression System in Camellia sinensis var. sinensis cv. ‘Tieguanyin’. Chinese Bulletin of Botany, 2022, 57(3): 340-349.
Treatment number | Cellulase (%) | Pectinase (%) | Macerozy-me (%) | Mannitol (mol·L-1) |
---|---|---|---|---|
1 | 1.4 | 0.3 | 0.1 | 0.3 |
2 | 1.4 | 0.4 | 0.2 | 0.4 |
3 | 1.4 | 0.5 | 0.3 | 0.5 |
4 | 1.5 | 0.3 | 0.2 | 0.5 |
5 | 1.5 | 0.4 | 0.3 | 0.3 |
6 | 1.5 | 0.5 | 0.1 | 0.4 |
7 | 1.6 | 0.3 | 0.3 | 0.4 |
8 | 1.6 | 0.4 | 0.1 | 0.5 |
9 | 1.6 | 0.5 | 0.2 | 0.3 |
表1 铁观音幼叶原生质体提取试剂配比
Table 1 Protoplast extraction reagent for Tieguanyin young leaves
Treatment number | Cellulase (%) | Pectinase (%) | Macerozy-me (%) | Mannitol (mol·L-1) |
---|---|---|---|---|
1 | 1.4 | 0.3 | 0.1 | 0.3 |
2 | 1.4 | 0.4 | 0.2 | 0.4 |
3 | 1.4 | 0.5 | 0.3 | 0.5 |
4 | 1.5 | 0.3 | 0.2 | 0.5 |
5 | 1.5 | 0.4 | 0.3 | 0.3 |
6 | 1.5 | 0.5 | 0.1 | 0.4 |
7 | 1.6 | 0.3 | 0.3 | 0.4 |
8 | 1.6 | 0.4 | 0.1 | 0.5 |
9 | 1.6 | 0.5 | 0.2 | 0.3 |
Reagent name | Amount (10 mL) |
---|---|
PEG-4000 | 4 g |
1 mol·L-1 CaCl2 | 2 mL |
0.8 mol·L-1 D-mannitol | 2.5 mL |
ddH2O | 2 mL |
表2 聚乙二醇(PEG)诱导铁观音原生质体转化试剂用量
Table 2 Reagent for polyethylene glycol (PEG)-induced Tieguanyin protoplast transformation
Reagent name | Amount (10 mL) |
---|---|
PEG-4000 | 4 g |
1 mol·L-1 CaCl2 | 2 mL |
0.8 mol·L-1 D-mannitol | 2.5 mL |
ddH2O | 2 mL |
Primer name | Primer sequence | Tm (°C ) |
---|---|---|
GS-F | TCCCCCGGGTCTCTTCTTTCCGATCTTTGCA | 66.3 |
GS-R | TCCCCCGGGTTACGGTTTCCAGAGGATGG | 68.1 |
TS-F | TCCCCCGGGGAGAAATTTGCAGAGCTGAGAG | 69.9 |
TS-R | TCCCCCGGGTCAATAGCGATGTATAAGTTGCTT | 64.4 |
表3 GSII-1.1和TSI基因引物序列
Table 3 Primer sequences of GSII-1.1 and TSI genes
Primer name | Primer sequence | Tm (°C ) |
---|---|---|
GS-F | TCCCCCGGGTCTCTTCTTTCCGATCTTTGCA | 66.3 |
GS-R | TCCCCCGGGTTACGGTTTCCAGAGGATGG | 68.1 |
TS-F | TCCCCCGGGGAGAAATTTGCAGAGCTGAGAG | 69.9 |
TS-R | TCCCCCGGGTCAATAGCGATGTATAAGTTGCTT | 64.4 |
Treatment number | Yield (106·g-1 FW) | Viability (%) |
---|---|---|
1 | 1.58±1.22 | 40.69±29.05 |
2 | 1.95±0.77 | 80.53±1.74 |
3 | 0.38±0.04 | 65.31±14.40 |
4 | 0.66±0.33 | 65.49±10.50 |
5 | 1.54±0.76 | 64.16±6.39 |
6 | 2.04±1.01 | 75.38±2.56 |
7 | 0.60±0.31 | 71.06±15.20 |
8 | 2.09±0.70 | 72.01±12.55 |
9 | 1.83±0.51 | 70.19±2.09 |
表4 不同酶解液配方下铁观音幼叶原生质体产量与活性(平均值±标准差)
Table 4 The number and viability of protoplasts extracted from young leaves of Tieguanyin under different enzymatic hydrolysis solutions (means±SD)
Treatment number | Yield (106·g-1 FW) | Viability (%) |
---|---|---|
1 | 1.58±1.22 | 40.69±29.05 |
2 | 1.95±0.77 | 80.53±1.74 |
3 | 0.38±0.04 | 65.31±14.40 |
4 | 0.66±0.33 | 65.49±10.50 |
5 | 1.54±0.76 | 64.16±6.39 |
6 | 2.04±1.01 | 75.38±2.56 |
7 | 0.60±0.31 | 71.06±15.20 |
8 | 2.09±0.70 | 72.01±12.55 |
9 | 1.83±0.51 | 70.19±2.09 |
图1 采用9号酶解液配方提取的铁观音幼叶原生质体 (A) 原生质体明场图; (B) 原生质体二乙酸荧光素染色图。Bars=50 μm
Figure 1 Tieguanyin young leaves protoplasts extracted using No. 9 enzymatic hydrolysis solution (A) Bright field image of protoplasts; (B) Fluorescein diacetate staining image. Bars=50 μm
图2 采用聚乙二醇(PEG)诱导HBT-TCP20-GFP质粒在拟南芥和铁观音叶肉原生质体中转化 (A)-(D) Bars=200 μm
Figure 2 Polyethylene glycol (PEG) induced transformation of HBT-TCP20-GFP plasmid into Arabidopsis thaliana and Tieguanyin mesophyll protoplasts (A)-(D) Bars=200 μm
图3 拟南芥和铁观音叶肉原生质体转化率 (A) HBT-GFP-GSII-1.1的转化率; (B) HBT-GFP-TSI的转化率。 *代表显著性差异水平(P<0.05, 卡方检验)。误差线表示平均值±标准差。
Figure 3 Transformation rate in Arabidopsis thaliana and Tieguanyin mesophyll protoplasts (A) Transformation rate of HBT-GFP-GSII-1.1; (B) Transformation rate of HBT-GFP-TSI. * represents significant difference (P<0.05, X2 test). Error bars represent means ± SD.
图4 拟南芥和铁观音叶肉原生质体中GSII-1.1和TSI蛋白亚细胞定位 (A)-(D) 拟南芥原生质体中转化HBT-GFP-GSII-1.1; (E)-(H) 拟南芥原生质体中转化HBT-GFP-TSI; (I)-(L) 铁观音叶肉原生质体中转化HBT-GFP-GSII-1.1; (M)-(P) 铁观音叶肉原生质体中转化HBT-GFP-TSI。Bars=10 μm
Figure 4 GSII-1.1 and TSI subcellular localization in Arabidopsis thaliana and Tieguanyin mesophyll protoplasts (A)-(D) Transformation of HBT-GFP-GSII-1.1 in Arabidopsis thaliana protoplasts; (E)-(H) Transformation of HBT-GFP-TSI in Arabidopsis thaliana protoplasts; (I)-(L) Transformation of HBT-GFP-GSII-1.1 in Tieguanyin protoplasts; (M)-(P) Transformation of HBT-GFP-TSI in Tieguanyin protoplasts. Bars=10 μm
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