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

doi:10.11983/CBB21206

Abstract

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

Yuqin Zhang, Jiacheng Wu, Meng He, Renyi Liu, Xiaoyue Zhu. An Efficient Protoplast Transient Expression System in Camellia sinensis var. sinensis cv. ‘Tieguanyin’[J]. Chinese Bulletin of Botany, 2022, 57(3): 340-349.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21206

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

Figures/Tables 8

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

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

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

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

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

Figure 2 Polyethylene glycol (PEG) induced transformation of HBT-TCP20-GFP plasmid into Arabidopsis thaliana and Tieguanyin mesophyll protoplasts (A)-(D) Bars=200 μm

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.

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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