松叶猪毛菜叶片原生质体的制备及优化

doi:10.13866/j.azr.2023.04.14

Abstract

Abstract:

The release of high-quality protoplasts from plant tissues is a prerequisite for the establishment of other technical systems such as protein subcellular localization, transient expression analysis, and protein-protein interactions. To quickly explore the special functional genes of the C₃-C₄ intermediate plant Salsola laricifolia, it is essential to establish a simple and efficient method for preparing leaf protoplasts. In this study, the euphylla of axenic tissue culture seedlings of S. laricifolia was used as material to analyze the effects of different concentration ratios of cellulase and isolated enzymes and osmotic pressure on protoplast isolation. The results showed that the true leaves of sterile tissue culture seedlings with a seedling age of 25 days were used for enzymatic hydrolysis in enzymatic hydrolysis solution of 2% cellulase + 0.5% isolated enzyme R-10 + 0.6 mol·L^-1 mannitol at 25 °C for 2 h, and W5 The solution was purified at a speed of 800 rpm·min^-1, the yield of protoplasts could reach 1.21 × 10⁶, and the viability was 85%. Using the obtained protoplast of S. laricifolia as a receptor, the pBI121-SaNADP-ME4-GFP plasmid vector was successfully transformed by the PEG transformation method, and it was detected that SaNADP-ME4 was located in the chloroplast. In this study, an efficient system for preparing protoplasts from the leaves of S. laricifolia was established, which lays the foundation for mining the special gene functions of this species.

Key words: Salsola laricifolia, leaf, protoplast, C₃-C₄ intermediate plant

XI Yuxin, ZHANG Yuhui, WEN Zhibin. Preparation and optimization of leaf protoplasts of Salsola laricifolia[J].Arid Zone Research, 2023, 40(4): 655-662.

Figures/Tables 5

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References 42

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编号	因素
编号	纤维素酶浓度/%	离析酶浓度/%	甘露醇浓度 /（mol·L^-1）	酶解时间/h
1	1.0	0.50	0.4	2
2	2.0	0.75	0.5	3
3	3.0	1.0	0.6	4

编号	纤维素酶浓度	离析酶浓度	甘露醇浓度 /(mol·L^-1)	酶解时间/h	原生质体产量 /[个·(100mg)^-1]	原生质体活力/%
1	1%	0.50%	0.4	2	(1.67±0.05)×10⁵	72.7±0.8
2	1%	0.75%	0.5	3	(4.07±0.18)×10⁵	85.2±0.9
3	1%	1.00%	0.6	4	(4.83±1.17)×10⁵	76.0±1.7
4	2%	1.00%	0.5	4	(4.67±0.13)×10⁵	73.3±1.4
5	2%	0.50%	0.6	2	(1.21±1.40)×10⁶	85.0±0.4
6	2%	0.75%	0.4	3	(6.97±0.18)×10⁵	79.5±1.1
7	3%	1.00%	0.6	3	(5.50±0.50)×10⁵	83.0±1.1
8	3%	0.50%	0.4	4	(2.88±0.38)×10⁵	74.3±1.8
9	3%	0.75%	0.5	2	(6.99±0.24)×10⁵	74.4±1.7
K1	1.06×10⁶	1.61×10⁶	1.15×10⁶	2.08×10⁶
K2	2.38×10⁶	1.80×10⁶	1.57×10⁶	1.65×10⁶
K3	1.54×10⁶	1.50×10⁶	2.250×10⁶	1.23×10⁶
k1	2.34×10⁶	2.32×10⁶	2.27×10⁶	2.32×10⁶
k2	2.38×10⁶	2.39×10⁶	2.33×10⁶	2.48×10⁶
k3	2.32×10⁶	2.32×10⁶	2.44×10⁶	2.24×10⁶
R	4.41×10⁵	1.01×10⁵	3.66×10⁵	2.81×10⁵
K1′	2.34	2.32	2.27	2.32
K2′	2.38	2.39	2.33	2.48
K3′	2.32	2.32	2.44	2.24
k1′	0.78	0.77	0.76	0.77
k2′	0.79	0.8	0.78	0.83
k3′	0.77	0.77	0.81	0.75
R′	0.02	0.02	0.06	0.08

Preparation and optimization of leaf protoplasts of Salsola laricifolia

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