铁观音原生质体高效瞬时转化方法的建立

doi:10.11983/CBB21206

植物学报 ›› 2022, Vol. 57 ›› Issue (3): 340-349.DOI: 10.11983/CBB21206

铁观音原生质体高效瞬时转化方法的建立

张玉琴¹^,², 吴嘉诚¹^,², 何萌², 刘仁义³, 朱晓玥²^,^*()

¹福建农林大学园艺学院, 福州 350002
²福建农林大学海峡联合研究院园艺生物学及代谢组学联合中心, 福州 350002
³福建农林大学海峡联合研究院农林生物大数据中心, 福州 350002

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:
福建农林大学导师研究经费(2018);国家自然科学基金(31970336);福建农林大学科技创新专项基金(CXZX2020094A);福建省自然科学基金(2021J01140)

An Efficient Protoplast Transient Expression System in Camellia sinensis var. sinensis cv. ‘Tieguanyin’

Yuqin Zhang¹^,², Jiacheng Wu¹^,², Meng He², Renyi Liu³, Xiaoyue Zhu²^,^*()

¹College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
²FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Fujian Agriculture and Forestry University, Fuzhou 350002, China
³Center for Agroforestry Mega Data Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Received:2021-11-24 Accepted:2022-02-07 Online:2022-05-01 Published:2022-05-18
Contact: Xiaoyue Zhu

摘要/Abstract

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。

关键词: 铁观音, 茶氨酸, 茶氨酸合成酶, 谷氨酰胺合成酶, 原生质体瞬时转化

Abstract: Recent advances in the genomic sequencing of tea plants have laid the foundation for tea research at the molecular and gene levels. However, the transgenic technologies are immature and the life cycles are long for tea trees, it is still difficult to conduct functional analyses of tea genes. This study used young leaves of Camellia sinensis var. sinensis cv. ‘Tieguanyin’, established a useful formula by testing multiple concentration combinations of cellulase, pectinase, macerozyme and mannitol. By evaluating the quantity, viability and debris of resulted protoplast, we successfully established a highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in Tieguanyin seedling leaves, with a transformation rate reaching 56.25%. Using this system, the subcellular localization of two pivotal enzymes in the theanine metabolism pathway (the theanine synthetase (TSI) and the glutamine synthetase (GSII-1.1)) were explored. Results show that, these two enzymes are both localized in the cytosol of the Tieguanyin protoplasts. Together, the establishment of this tea mesophyll protoplast extraction and transient expression system would lay a technological foundation for studying the function of tea genome.

Key words: Tieguanyin, theanine, theanine synthetase, glutamine synthetase, protoplast transfection

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.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21206

https://www.chinbullbotany.com/CN/Y2022/V57/I3/340

图/表 8

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

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

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

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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铁观音原生质体高效瞬时转化方法的建立

An Efficient Protoplast Transient Expression System in Camellia sinensis var. sinensis cv. ‘Tieguanyin’

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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

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

[1]	张智猛, 万书波, 宁堂原, 戴良香. 氮素水平对花生氮素代谢及相关酶活性的影响[J]. 植物生态学报, 2008, 32(6): 1407-1416.
[2]	黄其满, 刘伟华, 孙辉, 邓新, 苏金. 农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究 (英文)[J]. 植物生态学报, 2005, 29(2): 338-344.

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

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

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

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