干旱区研究

干旱区研究 >

2023 , Vol. 40 >Issue 4: 655 - 662

DOI: https://doi.org/10.13866/j.azr.2023.04.14

植物生态

松叶猪毛菜叶片原生质体的制备及优化

  • 席昱欣 ,
  • 张玉慧 ,
  • 闻志彬
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  • 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
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

收稿日期: 2022-07-07

  修回日期: 2022-12-12

  网络出版日期: 2023-04-28

基金资助

国家自然科学基金项目(31970354)

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Preparation and optimization of leaf protoplasts of Salsola laricifolia

  • Yuxin XI ,
  • Yuhui ZHANG ,
  • Zhibin WEN
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  • 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 date: 2022-07-07

  Revised date: 2022-12-12

  Online published: 2023-04-28

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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

本文引用格式

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 . DOI: 10.13866/j.azr.2023.04.14

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

参考文献

[1] Davey M R, Anthony P, Power J B, et al. Plant protoplasts: status and biotechnological perspectives[J]. Biotechnology Advances, 2005, 23(2): 131-171.
[2] 常胜合, 孙威, 许桂莺, 等. 植物原生质体分离方法及其应用研究进展[J]. 分子植物育种, 2018, 16(4): 1271-1277.
[2] [Chang Shenghe, Sun Wei, Xu Guiying, et al. Isolating method of plant protoplast and its research advances of application[J]. Molecular Plant Breeding, 2018, 16(4): 1271-1277.]
[3] Marion J, Bach L, Bellec Y, et al. Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings[J]. Plant Journal, 2008, 56(1): 169-179.
[4] Ren R, Gao J, Lu C Q, et al. Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in cymbidium orchids[J]. International Journal of Molecular Sciences, 2020, 21(7): 2264.
[5] Gorska A M, Gouveia P, Borba A R, et al. ZmOrphan94 transcription factor downregulates ZmPEPC1 gene expression in maize bundle sheath cells[J]. Frontiers in Plant Science, 2021, 12: 559-567.
[6] 王飞, 钟雄辉, 陈登辉, 等. 甘蓝原生质体制备体系优化及瞬时转化体系的建立[J]. 华北农学报, 2020, 35(3): 69-78.
[6] [Wang Fei, Zhong Xionghui, Chen Denghui, et al. Optimization of protoplasts preparation system and establishment of ttransient transformation system in Brassica oleracea var. capitata L.[J]. Acta Agricultuare Boreali-Sinica, 2020, 35(3): 69-78.]
[7] Pasternak T, Paponov I A, Kondratenko A, et al. Optimizing protocols for arabidopsis shoot and root protoplast cultivation[J]. Plants-Basel, 2021, 10(2): 375.
[8] Jeong Y Y, Lee H Y, Kim S W, et al. Optimization of protoplast regeneration in the model plant Arabidopsis thaliana[J]. Plant Methods, 2021, 17(1): 21.
[9] 尚飞, 李咪咪, 李莉, 等. 烟草原生质体的制备和分析[J]. 河南师范大学学报(自然科学版), 2013, 41(3): 130-137.
[9] [Shang Fei, Li Mimi, Li Li, et al. Preparation and alalysis of tobacco protoplasts[J]. Journal of Henan Normal University(Natural Science Edition), 2013, 41(3): 130-137.]
[10] 聂琼, 刘仁祥, 徐如宏. 烟草原生质体分离纯化条件的优化[J]. 贵州农业科学, 2012, 40(9): 20-23.
[10] [Nie Qiong, Liu Renxiang, Xu Ruhong. Optimization of isolation and purification of tobacco protoplast[J]. Guizhou Agricultural Sciences, 2012, 40(9): 20-23.]
[11] Zhang Y, Su J, Duan S, et al. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes[J]. Plant Methods, 2011, 30(7):1746-1748.
[12] 宋爱华, 张文斌, 孙姝兰, 等. 非洲菊原生质体制备及瞬时转化系统的建立[J]. 植物学报, 2017, 52(4): 511-519.
[12] [Song Aihua, Zhang Wenbin, Sun Shulan, et al. Preparation of protoplast and establishment of transient expression system in grebera hybrida[J]. Chinese Bulletin of Botany, 2017, 52(4): 511-519.]
[13] 姜倩倩, 陈磊, 李正男, 等. 多年生黑麦草原生质体制备及瞬时表达体系的建立[J]. 分子植物育种, 2021, 19(9): 2941-2948.
[13] [Jiang Qianqian, Chen Lei, Li Zhengnan, et al. Protoplast isolation and establishment of gene transient expression system in Lolium perenne L.[J]. Molecular Plant Breeding, 2021, 19(9): 2941-2948.]
[14] 仝亚楠, 刘雪, 刘秀洁, 等. 苋菜原生质体和瞬时转化体系的建立及应用[J]. 分子植物育种, 2021, 19(19): 6476-6481.
[14] [Tong Yanan, Liu Xue, Liu Xiujie, et al. Establishment and application of protoplast and transient transformation system of Edible amaranth (Amaranthus spp.)[J]. Molecular Plant Breeding, 2021, 19(19): 6476-6481.]
[15] Xu X F, Zhu H Y, Ren Y F, et al. Efficient isolation and purification of tissue-specific protoplasts from tea plants (Camellia sinensis (L.) O. Kuntze)[J]. Plant Methods, 2021, 17(1): 84.
[16] 李青, 鱼海鹏, 张子豪, 等. 棉花真叶原生质体分离及瞬时表达体系的优化[J]. 中国农业科学, 2021, 54(21): 4514-4524.
[16] [Li Qing, Yu Haipeng, Zhang Zihao, et al. Optimization of cotton mesophyll protoplast transient expression system[J]. Scientia Agricultura Sinica, 2021, 54(21): 4514-4524.]
[17] 王一菲, 刘新星, 张青, 等. 油棕叶肉原生质体分离及瞬时转化体系的建立[J]. 华中农业大学学报, 2021, 40(1): 154-159.
[17] [Wang Yifei, Liu Xinxing, Zhang Qing, et al. Isolation of oil palm mesophyll protoplasts and establishment of transient transformation system[J]. Journal of Huazhong Agricultural University, 2021, 40(1): 154-159.]
[18] 舒小娟, 温腾建, 邢佳毅, 等. 葡萄原生质体分离及瞬时转化体系的建立[J]. 西北植物学报, 2015, 35(6): 1262-1268.
[18] [Shu Xiaojuan, Wen Tengjian, Xing Jiayi, et al. Isolation of protoplast and establishment of transient transformation system in Grapevine(Vitisv inifera L.)[J]. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(6): 1262-1268.]
[19] Yao L P, Liao X, Gan Z Z, et al. Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)[J]. Scientia Horticulturae, 2016, 209: 14-21.
[20] Marion J, Bach L, Bellec Y, et al. Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of arabidopsis seedlings[J]. Plant Journal, 2008, 56(1): 169-179.
[21] Wang J J, Wang Y, Wang Y Z, et al. An efficient and universal protoplast isolation protocol suitable for transient gene expression analysis and single-cell RNA sequencing[J]. International Journal of Molecular Sciences, 2022, 23(7): 3419.
[22] 新疆植物志编辑委员会. 新疆植物志 (第二卷): 第一分册[M]. 乌鲁木齐: 新疆科技卫生出版社, 1994: 86-91.
[22] [Editorial Committee of Xinjiang Flora. Flora of Xinjiang (Volume II): Fascicle I[M]. Urumqi: Xinjiang Science and Technology Health Publishing House, 1994: 86-91.]
[23] Voznesenskaya E V, Koteyeva N K, Akhani H, et al. Structural and physiological analyses in Salsoleae (Chenopodiaceae) indicate multiple transitions among C3, intermediate, and C4 photosynthesis[J]. Journal of Experimental Botany, 2013, 64(12): 3583-3604.
[24] Wen Z B, Zhang M L. Salsola laricifolia, another C3-C4 intermediate species in tribe Salsoleae s.l. (Chenopodiaceae)[J]. Photosynthesis Research, 2015, 123(1): 33-43.
[25] Christin P A, Sage T L, Edwards E J, et al. Complex evolutionary transitions and the significance of C3-C4 intermediate forms of photosynthesis in molluginaceae[J]. Evolution, 2011, 65(3): 643-660.
[26] Sage R F, Khoshravesh R, Sage T L. From proto-kranz to C4 kranz: building the bridge to C4 photosynthesis[J]. Journal of Experimental Botany, 2014, 65(13): 3341-3356.
[27] Wen Z B, Wang Y L, Xia C L, et al. Chloroplastic SaNADP-ME4 of C3-C4 woody desert species Salsola laricifolia confers drought and salt stress resisance to Arabidopsis[J]. Plants-Basel, 2021, 10(9): 1827.
[28] 陈鹏. 小议血球计数板的使用[J]. 中学生物学, 2009, 25(8): 58-59.
[28] [Chen Peng. Discussion on the use of blood cell counting chamber[J]. Middle School Biology, 2009, 25(8): 58-59.]
[29] Wu F Q, Hanzawa Y. A simple method for isolation of soybean protoplasts and application to transient gene expression analyses[J]. Jove-Journal of Visualized Experiments, 2018, 131: 57258.
[30] 赵苏州, 卢运明, 张占路, 等. 玉米和拟南芥的原生质体制备及瞬时表达体系的研究[J]. 安徽农业科学, 2014, 42(12): 3479-3482.
[30] [Zhao Suzhou, Lu Yunming, Zhang Zhanlu, et al. Protoplast preparation and transient expression system in maize and Arabidopsis[J]. Journal of Anhui Agricultural Sciences, 2014, 42(12):3479-3482.]
[31] 沈雁翔, 梁言, 王鹏, 等. 舞春花原生质体分离条件的优化研究[J]. 上海农业科技, 2022(2): 15-18.
[31] [Shen Yanxiang, Liang Yan, Wang Peng, et al. Study on the optimization of protoplast separation conditions of Calibrachoa hybrida[J]. Shanghai Agricultural Science and Technology, 2022(2): 15-18.]
[32] 陈名红, 熊立, 陈学军. 烟草叶肉原生质体分离和纯化研究[J]. 云南民族大学学报(自然科学版), 2005, 24(4): 326-329.
[32] [Chen Minghong, Xiong Li, Chen Xuejun. Isolation and purification of mesophyll protoplast in tabacco[J]. Journal of Yunnan Minzu University(Natural Sciences Edition), 2005, 24(4): 326-329.]
[33] 谢鑫, 蒋君梅, 王勇, 等. 高粱原生质体的制备及转化方法研究[J]. 种子, 2019, 38(8): 43-46.
[33] [Xie Xin, Jiang Junmei, Wang Yong, et al. Study on the method of protoplast isolation and transformation of Sorghumbicolor[J]. Seed, 2019, 38(8): 43-46.]
[34] 赖叶林, 贺莹, 李欣欣, 等. 一种植物原生质体分离与瞬时转化的方法[J]. 植物生理学报, 2020, 56(4): 895-903.
[34] [Lai Yelin, He Ying, Li Xinxin, et al. An approach to isolation and transient transformation of protoplasts in plants[J]. Plant Physiology Journal, 2020, 56(4): 895-903.]
[35] 楠迪娜, 薛敏, 唐宽刚, 等. 沙冬青子叶原生质体瞬时表达体系的建立及其AmDREB1蛋白的亚细胞定位[J]. 植物科学学报, 2018, 36(4): 562-568.
[35] [Nan Dina, Xue Min, Tang Kuangang, et al. Establishment of the cotyledon protoplast transient expression system of Ammopiptanthus mongolicus and subcellular localization of the AmDREB1 protein[J]. Plant Science Journal, 2018, 36(4): 562-568.]
[36] 谷战英, 杨若楠, 陈昊. 油桐叶肉细胞原生质体分离及瞬时转化体系的建立[J]. 林业科学, 2018, 54(1): 46-53.
[36] [Gu Zhanying, Yang Ruonan, Chen Hao. The establishment of isolation and transient transformation methods of protoplass of Vernicia fordii mesophyll cells[J]. Scientia Silvae Sinicae, 2018, 54(1): 46-53.]
[37] 李婧瑶, 刘龙飚, 丁兵, 等. 植物原生质体分离及培养研究进展[J]. 分子植物育种, 2021, 21(2): 620-632.
[37] [Li Jingyao, Liu Longbiao, Ding Bing, et al. Research progress on isolation and culture of plant protoplasts[J]. Molecular Plant Breeding, 2021, 21(2): 620-632.]
[38] Zhou J, Wang B C, Zhu L Q. Conditioned culture for protoplasts isolated from chrysanthemum: An efficient approach[J]. Colloids and Surfaces B-Biointerfaces, 2005, 45(3-4): 113-119.
[39] 周波, 聂玉哲, 张晓磊, 等. 芜菁原生质体的分离及绿色荧光蛋白的瞬时表达[J]. 生物技术通讯, 2008(4): 542-544.
[39] [Zhou Bo, Nie Yuzhe, Zhang Xiaolei, et al. Protoplast isolation of brassica rapa ‘tsuda’ turnip and transient expression of green fluor escent protein[J]. Letters in Biotechnology, 2008(4): 542-544.]
[40] 杜小云, 王震, 王翠华, 等. “红颜”草莓原生质体制备及瞬时转化体系的建立[J]. 生物资源, 2019, 41(6): 532-538.
[40] [Du Xiaoyun, Wang Zhen, Wang Cuihua, et al. Preparation of protoplasts and establishment of transient transformation system of Hongyan strawberry[J]. Biotic Resources, 2019, 41(6): 532-538.]
[41] Chamani E, Tahami S K, Zare N, et al. Effect of different cellulase and pectinase enzyme treatments on protoplast isolation and viability in lilium ledebeourii bioss[J]. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2012, 40(2): 123-128.
[42] 雷海英, 白凤麟, 冯宇, 等. 玉米叶片原生质体的制备及瞬时转化体系的建立[J]. 长治学院学报, 2018, 35(2): 11-14.
[42] [Lei Haiying, Bai Fenglin, Feng Yu, et al. Preparation of maize leaf protopasts and establishment of transient transformation system[J]. Journal of Changzhi University, 2018, 35(2): 11-14.]
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