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

干旱区研究 ›› 2023, Vol. 40 ›› Issue (4): 655-662.doi: 10.13866/j.azr.2023.04.14 cstr: 32277.14.AZR.20230414

松叶猪毛菜叶片原生质体的制备及优化

席昱欣^1,^2,³(),张玉慧^1,^3,⁴,闻志彬^1,^2,^4,⁵()

1.中国科学院新疆生态与地理研究所,荒漠与绿洲生态国家重点实验室，新疆乌鲁木齐 830011
2.新疆抗逆植物基因资源保育与利用重点实验室，新疆乌鲁木齐 830011
3.中国科学院大学，北京 100049
4.中国-塔吉克斯坦生物资源保育与利用联合实验室，新疆乌鲁木齐 830011
5.中国科学院新疆生态与地理研究所标本馆，新疆乌鲁木齐 830011

收稿日期:2022-07-07 修回日期:2022-12-12 出版日期:2023-04-15 发布日期:2023-04-28
作者简介:席昱欣（1999-），女，在读研究生，研究方向为植物抗逆分子生物学. E-mail: xiyuxin20@mails.ucas.ac.cn
基金资助:
国家自然科学基金项目(31970354)

Preparation and optimization of leaf protoplasts of Salsola laricifolia

XI Yuxin^1,^2,³(),ZHANG Yuhui^1,^3,⁴,WEN Zhibin^1,^2,^4,⁵()

1. State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academyof Sciences, Urumqi 830011, Xinjiang, China
2. Xinjiang Key Laboratory of Stress to Tolerance Plant Resources Conservation and Resistance Gene Utilization, Urumqi 830011, Xinjiang, China
3. University of Chinese Academy of Sciences, Beijing 100049, China
4. Sino-Tajikistan Joint Laboratory for Conservation and Utilization of Biological Resources, Urumqi 830011, Xinjiang, China
5. The Specimen Museum of Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, Xinjiang, China

Received:2022-07-07 Revised:2022-12-12 Published:2023-04-15 Online:2023-04-28

摘要/Abstract

摘要：

从植物组织中释放出高质量的原生质体是建立其他技术体系如蛋白亚细胞定位、瞬时表达分析、蛋白质间互作的前提。为了快速挖掘C₃-C₄中间型植物松叶猪毛菜（Salsola laricifolia）的特殊功能基因，建立一个简单高效的叶片原生体的制备方法必不可少。本研究以松叶猪毛菜无菌组培苗的真叶为材料，分析不同纤维素酶和离析酶的浓度配比、渗透压对原生质体分离的影响。结果表明：采用25 d龄的无菌组培苗真叶，在2%纤维素酶+0.5%离析酶+0.6 mol·L^-1甘露醇的酶解液中25 ℃酶解2 h，使用W5溶液在800 rpm·min^-1的转速下纯化，原生质体产量可达1.21×10⁶个，活力为85%。并且利用得到的松叶猪毛菜原生质体作为受体，用PEG转化法成功转化pBI121-SaNADP-ME4-GFP质粒载体，检测到SaNADP-ME4定位于叶绿体中。本项研究建立了松叶猪毛菜叶片原生质体高效制备体系，为松叶猪毛菜特殊基因功能的挖掘奠定了基础。

关键词: 松叶猪毛菜(Salsola laricifolia), 叶片, 原生质体, C₃-C₄中间型植物

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

席昱欣, 张玉慧, 闻志彬. 松叶猪毛菜叶片原生质体的制备及优化[J]. 干旱区研究, 2023, 40(4): 655-662.

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

图/表 5

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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

松叶猪毛菜叶片原生质体的制备及优化

Preparation and optimization of leaf protoplasts of Salsola laricifolia

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