植物原生质体制备及转化试剂盒                               

●  产品组成：

● 产品简介：

植物原生质体是指脱去全部细胞壁由质膜包被的具有生命活力的裸露细胞。它具有细胞生命特征和全能型，是细胞无性系变异和突变体筛选的重要来源，同时也是植物遗传工程的理想受体和遗传改良的理想材料。酶解法分离原生质体是一个常用的技术，其原理是植物细胞壁主要由纤维素、半纤维素和果胶质组成，因而使用纤维素酶、半纤维素酶、离析酶和果胶酶能降解细胞壁成分，去除细胞壁，即可得到原生质体。许多方法可诱导原生质体融合，现在被广泛采用的融合方法是聚乙二醇（PEG）法。聚乙二醇（PEG）作为一种高分子化合物，合适的浓度能对原生质体产生瞬间冲击效应，原生质体很快发生收缩与粘连，随后用高钙高pH法进行清洗，使原生质体融合得以完成。

按照每次使用5 ml酶消化体系计算，本试剂盒可用于40次酶消化反应。本试剂盒每个5 ml反应体系可处理0.1 g左右的拟南芥叶片(约10~15叶片)，操作较好的情况下每5 ml体系可获得约50-70万个原生质体(不同植物不同操作会有一定的差异)，可满足约25-70个样品的原生质体质粒转染操作(按照每样1-2万个细胞计算)。

原生质体转化时，本试剂盒可以用于相当于6孔板40个孔的样品，12孔板80个孔的样品，或24孔板160个孔的样品的转化。

● 贮存和效期：

-20°C保存，至少一年有效。

组分I，II，III，V 4℃存放3-5天不会影响使用效果，长期不用，按照标签-20℃贮存。

试剂盒常温运输。

● 使用说明：

   需要准备的材料（试剂盒不提供）：

   剪尖吸头（剪尖后可以用酒精灯过火将吸头处理平滑）；纤维素酶R-10；离析酶R-10；平头镊子；70μm细胞过滤筛；一次性刀片；50ml离心管；1.5ml离心管；水浴锅。

一、原生质体分离：

即用型酶溶液配制

注：即用型酶溶液现用现配，不建议配制后冻存后再使用；

溶解好的正常酶溶液应为澄清棕黄色溶液，如使用纯度不好的酶，溶液为不溶解的乳白色悬浊液，不能使用。

1. 酶解：

取生长状态良好的叶片切成0.5-1 mm的小条，按照0.1 克叶片（拟南芥约15-20个叶片）加5 ml即用型酶溶液的比例迅速将切割好的叶片小条浸泡于酶溶液中，避光，无需震荡，常温（20-25℃）酶解3小时，间歇混匀。

注：酶解时间与叶片种类，叶片生长状态有关，请根据实验需要适当调整酶解时间。3小时为拟南芥叶片推荐的酶解时间。如条件允许，可以使用微型真空泵常温避光条件下抽真空30 min，以使酶溶液更好地进入细胞间隙。酶溶液变为绿色表明有原生质体已经有分离，溶液为浓绿色表明原生质体已经大量分离。拟南芥原生质体大小约为30-50 μm，显微镜镜检后确定是否分离。叶片原生质体细胞显微镜下为绿色圆球状，叶绿体分散在整个细胞内，说明状态较好；如呈现不规则形状，说明原生质体破碎或即将破碎。

2. 漂洗：

酶解后加入等体积的溶液II，如使用5 ml酶解体系，加入5 ml溶液II，轻柔混匀。

3. 过滤：

用孔径70 μm筛网过滤步骤2中的溶液，去除未消化的叶片，收集滤出液于50 ml离心管中。

4. 第一次收集：

滤出液100g常温离心 2分钟，尽量去除上清。

注：为了避免原生质体离心时贴在管壁，建议整个实验过程使用水平转头；离心时，可调低离心机的升速和降速。升速过快，原生质体可能离到管壁上；降速过快，可能导致管底原生质体悬起。

5. 第二次收集：

加入2-5 ml 溶液II，用剪尖的蓝吸头重悬原生质体，冰浴30分钟，原生质体在重力作用下可以沉降到离心管底部，尽量吸除上清，收集原生质体。(如果发现原生质体沉降的速度比较慢或者得率比较低，也可以考虑常温100 g离心1-2 min收集原生质体)。

6. 重悬：

小心去除上清溶液，不要触动原生质体沉淀，沉淀用剪尖的蓝吸头重悬于1 ml溶液III中即为原生质体溶液。（可以用血球计数板计数，根据原生质体数量调整溶液III的加入体积，使得原生质体密度为2×10⁵/ml或更高）。

注：制备好的原生质体可以在4°C或冰浴保存至少24 h。

二、原生质体转化和培养：

  1. 转化溶液配制：

植物原生质体转化参考下表根据样品量配制转化溶液。

转化溶液现用现配。转化溶液需要在转化前至少1小时配制，以确保转化试剂溶解充分。转化溶液配制后尽量当天使用。配制好的转化溶液4℃保存3-5天之内仍然有较好的转化效率，但和当天配制的转化溶液相比，转化效果可能会有一定程度的下降。

2. 准备质粒：

取10 μl质粒DNA(10-20 μg，浓度为1-2 μg/μl)于1.5 ml 离心管中。

注：质粒大小建议为5-10 kb，质粒浓度为1-2 μg/μl左右；

原生质体转化对于质粒的纯度要求较高，尽量使用高纯度的质粒。

3. 质粒转化：

3.1 质粒管中加入100 μl原生质体溶液（原生质体密度为2×10⁵/ml，约2万个原生质体），轻柔混匀。

3.2 加入等体积即110 μl 步骤1事先准备好的转化溶液，轻弹管底，轻柔混匀，常温25℃放置5-15分钟。

注：最长可以孵育15 min，但通常孵育5min时间已经足够。最佳的孵育时间对于不同的原生质体和不同的质粒需要通过实验摸索。

4. 终止转化：

加入2倍体积即440 μl 溶液II，轻柔彻底混匀，终止转化过程。

5. 收集原生质体：

常温100g离心1-2分钟，尽量去除上清。

注：由于转化后的溶液会非常粘稠，加入溶液II后离心1 min通常可以使原生质体聚集在管底，但使用某些突变体时为减少原生质体损失，可将离心时间延长到2 min。

6. 原生质体漂洗：

  加入500 μl溶液II轻轻重悬原生质体，常温100g离心1 min，尽量去除残留的上清。注：本步骤可以充分去除残留的转染试剂溶液中的组分，避免转染试剂溶液中的组分对于后续的不良影响。

7. 原生质体重悬：

沉淀中加入1 ml溶液V，轻柔重悬。

8. 原生质体培养：

将离心管水平放置，23-25oC弱光培养。

注：根据实验需求确定孵育时间。RNA分析孵育2-6小时；酶活性分析和蛋白标记实验孵育2-16小时；基因编辑的效果在转染24小时后可能被检测到。

三、实验示例：

 使用原生质体植物原生质体制备及转化试剂盒转染拟南芥原生质体的效果图。

实验步骤：称取0.48 g转化试剂于2 ml 离心管中，加入转化试剂溶解液后，颠倒混匀，蒸馏水定容至2 ml，使转化试剂充分溶解后备用。在2 ml的圆底离心管中加入10 μl（20 μg）EGFP质粒 (植物用绿色荧光蛋白)，加入100 μl制备好的原生质体，轻柔混匀后加入110 μl当日配制好的转化试剂溶液，轻柔混匀，常温静置5 min后加入440 μl溶液II终止转化，轻轻颠倒混匀，常温100 g离心1 min，去除上清，再加入0.5 ml溶液II，轻柔重悬原生质体，常温100 g离心1 min后，尽量去除上清，收集原生质体。加入1 ml溶液V，小心重悬原生质体后水平放置25℃培养过夜(约16h)，次日于荧光显微镜下检测EGFP荧光信号。

纤维素酶R-10

离析酶R-10

果胶酶Y-23

一次性细胞过滤筛

不锈钢细胞过滤筛

植物原生质体提取试剂盒发表文章列表（27篇）

1. [IF=3.19] TaEXPB7-B,a -expansin gene involved in low-temperature stress and abscisic acid responses, promotes growth and cold resistance in Arabidopsis thaliana.

实验植物：小麦

Author: Xu Feng, Yongqing Xu, Lina Peng, Xingyu Yu, Qiaoqin Zhao, Shanshan Feng, Ziyi Zhao, Fenglan Li, Baozhong Hu.

Journal: J Plant Physiology 2019

Institution：  College of Life Sciences, Northeast Agricultural University

Paper link：http://www.plantphysiol.org/content/174/4/2487

2. [IF=5.36] Involvement of the chloroplast gene ferredoxin 1 in multiple responses of Nicotiana benthamiana to Potato virus X infection. 

实验植物：烟草

Author: Xue Yang, Yuwen Lu, Fang Wang, Ying Chen, Yanzhen Tian, Liangliang Jiang, Jiejun Peng, Hongying Zheng, Lin Lin, Chengqi Yan, Michael Taliansky, Stuart MacFarlane, Yuanhua Wu,  Jianping Chen and Fei Yan

Journal: Journal of Experimental Botany, 2020，Vol.71, No. 6,2142–2156, 

Institution：Institute of Plant Virology, Ningbo University 

Paper link：https://academic.oup.com/jxb/article/71/6/2142/5686178?login=true

3. [IF=7.228]  Turnip mosaic virus impairs perinuclear chloroplast clustering to facilitate viral infection

实验植物：烟草

Author:Yushan Zhai, Quan Yuan, Shiyou Qiu, Saisai Li, Miaomiao Li, Hongying Zheng, Guanwei Wu, Yuwen Lu, Jiejun Peng, Shaofei Rao, Jianping Chen, Fei Yan

Journal: Plant Cell Enviroment, 2021，30 July 

Institution：Institute of Plant Virology, Ningbo University 

Paper link：https://onlinelibrary.wiley.com/doi/10.1111/pce.14157

4. [IF=5.64]  A Novel, Small Cysteine-Rich Effector, RsSCR10 in Rhizoctonia solani Is Sufficient to Trigger Plant Cell Death

实验植物：水稻

Author:Xianyu Niu, Guijing Yang, Hui Lin, Yao Liu, Ping Li and Aiping Zheng

Journal: Fronties in Microbiology August 2021 | Volume 12 | Article 684923 

Institution：Sichuan Agricultural University

Paper link：https://www.frontiersin.org/articles/10.3389/fmicb.2021.684923/full

5. [IF=9.8]  Synthesis of flavour-related linalool is regulated by PpbHLH1 and associated with changes in DNA methylation during peach fruit ripening

实验植物：烟草

Author: Chunyan Wei, Hongru Liu, Xiangmei Cao, Minglei Zhang, Xian Li, Kunsong Chen and Bo Zhang

Journal: Plant Biotechnology Journal (2021) 19, pp. 2082–2096 

Institution：Laboratory of Fruit Quality Biology/Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Zhejiang University

Paper link：https://onlinelibrary.wiley.com/doi/10.1111/pbi.13638

6. [2020IF=1.04]  EoPHR2, a Phosphate Starvation Response Transcription Factor, Is Involved in Improving Low-Phosphorus Stress Resistance in Eremochloa ophiuroides

实验植物：拟南芥

Author: Ying Chen1,#, Chuanqiang Liu1,#, Qingqing He1, Jianjian Li2, Jingjing Wang2, Ling Li2, Xiang Yao2, Shenghao Zhou3, Haoran Wang

Journal: Phyton Vol.91, No.3, 2022, pp.651-665

Institution： Institute of Botany, Jiangsu Province and Chinese Academy of Sciences

Paper link：https://www.techscience.com/phyton/v91n3/45309

7. [2021IF=3.2]  Establishment and optimization of PEG-mediated protoplast transformation in the microalga Haematococcus pluvia

实验材料：雨生红球藻 Haematococcus pluvia

Author: Chunli Guo，Muhammad Anwar， Rui Mei，Xinyi Li， Di Zhao，Yanan Jiang， Jieyi Zhuang， Chen Liu，Chaogang Wang， Zhangli Hu

Journal: Journal of Applied Phycology. Published online 07 March 2022

Institution：College of Optoelectronic Engineering, Shenzhen University

Paper link： https://link.springer.com/article/10.1007/s10811-022-02718-x

8. [2021 IF=5.9] The Genome-Wide Identification of Long Non-Coding RNAs Involved in Floral Thermogenesis in Nelumbo nucifera Gaertn

实验材料：睡莲 Nelumbo nucifera Gaertn

Author: Jing Jin , Yu Zou, Ying Wang, Yueyang Sun, Jing Peng and Yi Ding

Journal: Int. J. Mol. Sci. 2022, 23, 4901.

Institution：College of Life Sciences, Guizhou University，College of Life Sciences, Wuhan University

Paper link： https://www.mdpi.com/1422-0067/23/9/4901

9. [2021 IF=17.9] Genome-wide association analysis reveals a novel pathway mediated by a dual-TIR domainprotein for pathogen resistance in cotton

实验材料：棉花

Author: Yihao Zhang, Yaning Zhang, Xiaoyang Ge, Yuan Yuan, Yuying Jin, Ye Wang, Lihong Zhao, Xiao Han, Wei Hu, Lan Yang, Chenxu Gao, Xi Wei, Fuguang Li, Zhaoen Yang

Journal: Genome Biology  (2023) 24:111

Institution：Institute of Cotton Research, Chinese Academy of Agricultura Sciences

Paper link： https://doi.org/10.1186/s13059-023-02950-9

10. [2022 IF=7.4] Rose long noncoding RNA lncWD83 promotes flowerin by modulating ubiquitination of the floral repressor RcMYC2L

实验材料：Rose 玫瑰

Author: Chen Yeqing ,Lu  Jun ,Wang Weinan ,Fan Chunguo ,Yuan Guozhen ,Sun  Jingjing ,Liu  Jinyi ,Wang Changquan

Journal: Plant Physiol (2023) Published: 19 September 2023

Institution：College of Horticulture, Nanjing Agricultural University

Paper link： https://doi.org/10.1093/plphys/kiad502

11. [2022 IF=17.4] Functionalized carbon nano-enabled plant ROS signal engineering for growth / defense balance

实验材料：拟南芥

Author: Zhijiang Guo , Qiong Chen , Taibo Liang , Baoyuan Zhou , Suhua Huang , Xiufeng Cao, Xiuli Wang , Zaisong Ding, Jiangping Tu

Journal: Nano Today 53 (2023) 102045

Institution：School of Materials Science and Engineering, Zhejiang University

Paper link：https://doi.org/10.1016/j.nantod.2023.102045

12. [2022 IF=13.8] Unveiling the mechanism of broad-spectrum blast resistance in rice: The collaborative role of transcription factor OsGRAS30 and histone deacetylase OsHDAC1

实验材料：水稻

Author: Jiaqi Hou, Huangzhuo Xiao, Peng Yao, Xiaoci Ma, Qipeng Shi, Jin Yang, Haoli Hou and Lijia Li

Journal: Plant Biotechnology Journal (2024), pp. 1–17

Institution：State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University

Paper link：https://doi.org/10.1111/pbi.14299

13. [2022 IF=7.2] NnSnRK1-NnATG1-mediated autophagic cell death governs flower bud abortion in shaded lotus

实验材料：荷花

Author: Xiehongsheng Li, Yingchun Xu, Zongyao Wei, Jiaying Kuang, Mingzhao She, Yanjie Wang and Qijiang Jin

Journal: Plant J (2024) 117, 979–998

Institution：College of Horticulture, Nanjing Agricultural University

Paper link： https://doi.org/10.1111/tpj.16655

14. [2022 IF=7.5] The dynamic TaRACK1B-TaSGT1-TaHSP90 complex modulates NLR-protein-mediated antiviral immunity in wheat.

实验材料：小麦

Author: Haichao Hu, Tianye Zhang, Jinnan Wang, Jun Guo, Yaoyao Jiang, Qiansheng Liao, Lu Chen, Qisen Lu,Peng Liu, Kaili Zhong, Jiaqian Liu, Jianping Chen, Jian Yang

Journal: Cell Reports 43, 114765, October 22, 2024

Institution：Institute of Plant Virology, Ningbo University

Paper link： https://doi.org/10.1016/j.celrep.2024.114765

15. [2023 IF=14.3] Natural SNP Variation in GbOSM1 Promotor Enhances Verticillium Wilt Resistance in Cotton.

实验材料：棉花

Author: Guilin Wang, Dayong Zhang, Haitang Wang, Jinmin Kong, Zhiguo Chen, Chaofeng Ruan, Chaoyang Deng, Qihang Zheng, Zhan Guo, Hanqiao Liu, Weixi Li, Xinyu Wang, Wangzhen Guo

Journal: Advanced Science 2024, 2406522

Institution：Nanjing Agricultural University

Paper link：https://doi.org/10.1002/advs.202406522

16. [2023 IF=14.7] Re-localization of a repeat-containing fungal effector by apoplastic protein Chitinase-like 1 blocks its toxicity.

实验材料：拟南芥

Author: Hanqiao Liu, Wenshu Zhang, QinqfeiHe, Reyila Aikemu,HuijuanXu, Zhan Guo, LuWang, WeixiLi, GuilinWang, XinyuWang , Wangzhen Guo

Journal: Nature Communications (2024)15:10122

Institution：Nanjing Agricultural University

Paper link：https://doi.org/10.1038/s41467-024-54470-0

17. [2023 IF=14.7] Rational design of phytovirucide inhibiting nucleocapsid protein aggregation in tomato spotted wilt virus.

实验材料：烟草

Author: Ningning Zan ,JiaoLi ,JiahuiYao , ShangWu ,JianzhuanLi , Feifei Chen,Baoan Song, Runjiang Song

Journal: Nature Communications  2025，16:2034

Institution：Guizhou University

Paper link：https://doi.org/10.1038/s41467-025-57281-z

18. [2023 IF=4.8] lncRNA- Encoded Small Peptide Promotes Viral Infection.

实验材料：烟草

Author: Yaoyao Li, Juan Zhang,Chunyan Qi,Qitao Mo,Kaili Zhong,Jiaqian Liu,Hailin Cai,Jianyong Li,Jianping Chen,Jian Yang,Peng Liu

Journal: Molecular Plant Pathology 2025; 26:e70084

Institution：Ningbo University

Paper link：https://doi.org/10.1111/mpp.70084

19. [2023 IF=6.5] OsHDAC1 deacetylates the aldehyde dehydrogenase OsALDH2B1, repressing OsGR3 and decreasing salt tolerance in rice

实验材料：水稻

Author: Yequn Wu, Jiaqi Hou, Huangzhuo Xiao, Shiqi Ye, Daoyi Tu, Ronghua Qiu, Xiaoci Ma, Yating Zhao, Tingyu Chen and Lijia Li

Journal: Plant Physiology, kiaf149，Published: 11 April 2025

Institution：Wuhan University

Paper link： https://doi.org/10.1093/plphys/kiaf149

20. [2023 IF=14.7] An m⁶A methyltransferase confers host resistance by degrading viral proteins through ubiquitination.

实验材料：小麦

Author: Jun Guo, Tianye Zhang, Haoxin Xie, Haichao Hu, Chaonan Shi, Yingjie Zhao,Jingliang Yin, Gecheng Xu, Zechi Wu, Pengkun Wang, Jiaqian Liu, Peng Liu,Kaili Zhong, Feng Chen, Jianping Chen，Jian Yang

Journal: Nature Communications ，(2025) 16:4821

Institution：Ningbo University

Paper link： https://doi.org/10.1038/s41467-025-60199-1

21. [2023 IF=4.8] Bioremediation potential of lowbrominated polybrominated diphenyl by the phyllospheric Wickerhamomyces anomalus.

实验材料：烟草

Author: JMan Cai, Zheng Xing, Xiaolei Yuan, Zhangyu Song,Xiaojing Wang，Kejiu Du

Journal: Frontiers in Plant Science  Published 11 July 2025

Institution：College of Forestry, Hebei Agricultural University

Paper link： https://doi.org/10.3389/fpls.2025.1606962

22. [2025 IF=6.9] ISOPENTENYL TRANSFERASE3 activation triggers root ammonium hypersensitivity via the cytokinin-ARR10/ARR12-CAP1 signaling pathway

实验材料：拟南芥

Author: Ting-Ting Li, Lei Wu, Meng Wang, Herbert J Kronzucker, Yunqi Liu, Weiming Shi , Dong-Wei Di

Journal: Plant Physiology 2025 Sep 29:kiaf462

Institution：Institute of Soil Science, Chinese Academy of Sciences

Paper link：https://doi.org/10.1093/plphys/kiaf462

23. [2025 IF=8.1] Rapid induction of NH₄⁺ efflux in rice roots under high-ammonium stress and its association with varietal differences in ammonium tolerance and ammonium-utilization efficiency.

实验材料：水稻

Author: Dong-Wei Di , Ting-Ting Li, Jingjing Wu,Meng Wang , Herbert J. Kronzucker , Yunqi Liu, Chuanfa Liu，Weiming Shi

Journal: New Phytologist (2025) 248: 1625–1638

Institution：Institute of Soil Science, Chinese Academy of Sciences

Paper link： https://doi.org/10.1111/nph.70590

24. [2025 IF=4.8] Bioremediation potential of lowbrominated polybrominated diphenyl by the phyllospheric Wickerhamomyces anomalus.

实验材料：烟草

Author: Man Cai, Zheng Xing, Xiaolei Yuan, Zhangyu Song,Xiaojing Wang，Kejiu Du

Journal: Frontiers in Plant Science (2025) Volume 16，2025

Institution：College of Forestry, Hebei Agricultural University

Paper link： https://doi.org/10.3389/fpls.2025.1606962

25. [2025 IF=7.1] Phosphorylation and ubiquitination coordinate homeostasis of a tomato transporter responsible for fruit sugar accumulation.

实验材料：烟草

Author: Bangqian Song, Yong Kang, Xi Zheng, Zhengguo Li, Yulin Cheng

Journal: Journal of Genetics and Genomics.  Accepted Date: 22 March 2026

Institution：School of Life Sciences, Chongqing University

Paper link：https://doi.org/10.1016/j.jgg.2026.03.019

26. [2025 IF=5.7] A Reciprocal Feedback Loop Between Growth-Regulating Factor 9 and SNF1-Related Protein Kinase 1 Integrates Shade Signaling with Autophagy-Mediated Flower Bud Abortion in Lotus.

实验材料：荷花

Author: Jiaying Kuang, Ziyu Chen, Kefan Liang, Yifan Li, Yanjie Wang, Yingchun Xu, Qijiang Jin

Journal: Journal of Experimental Botany 2026

Institution：College of Horticulture, Nanjing Agricultural University

Paper link：https://doi.org/10.1093/jxb/erag152

27. [2025 IF=6.9] A cis-regulatory variant in ZmABI45 modulates drought tolerance and adaptation by escaping ZmbHLH80 repression in maize.

实验材料：玉米

Author: Xuyang Liu, Yue Hu, Sen Xie, Keyu Tao, Youyu Zhao, Yaxuan Lu, Huaijun Tang, Yanjun Zhang, Yongxiang Li, Dengfeng Zhang, Guanhua He, Zhenju Li, Tianyu Wang, Chunhui Li, Yu Li

Journal: Cell Reports 45, 117334, May 26, 2026

Institution：Institute of Crop Sciences, Chinese Academy of Agricultural Sciences

Paper link：https://doi.org/10.1016/j.celrep.2026.117334