植物原生質(zhì)體制備試劑盒                                        

●  產(chǎn)品組成：

● 產(chǎn)品簡(jiǎn)介：

植物原生質(zhì)體是指脫去全部細胞壁由質(zhì)膜包被的具有生命活力的裸露細胞。它具有細胞生命特征和全能型，是細胞無(wú)性系變異和突變體篩選的重要來(lái)源，同時(shí)也是植物遺傳工程的理想受體和遺傳改良的理想材料。酶解法分離原生質(zhì)體是一個(gè)常用的技術(shù)，其原理是植物細胞壁主要由纖維素、半纖維素和果膠質(zhì)組成，因而使用纖維素酶、半纖維素酶、離析酶和果膠酶能降解細胞壁成分，去除細胞壁，即可得到原生質(zhì)體。試劑盒配套的酶混合物是纖維素酶R-10和離析酶R-10的混合物，優(yōu)化的酶配比能很好地消化植物細胞壁，提取到良好的植物原生質(zhì)體。

按照每次使用5 ml酶消化體系計算，本試劑盒可用于20次酶消化反應。本試劑盒每個(gè)5 ml反應體系可處理0.1 g左右的擬南芥葉片(約10~15葉片)，操作較好的情況下每5 ml體系可獲得約50-70萬(wàn)個(gè)原生質(zhì)體(不同植物不同操作會(huì )有一定的差異)，可滿(mǎn)足約25-70個(gè)樣品的原生質(zhì)體質(zhì)粒轉染操作(按照每樣1-2萬(wàn)個(gè)細胞計算)。

本試劑盒不含有原生質(zhì)體轉化試劑，需要轉化請參見(jiàn)植物原生質(zhì)體轉化試劑盒（RTU4092 ）。

● 貯存、效期及運輸：

  按照標簽溫度保存，至少一年有效。-20℃貯存組分4℃存放3-4不會(huì )影響使用效果。3-4天經(jīng)常使用的情況，可以全部保存在4℃。

試劑盒常溫運輸。

● 使用說(shuō)明：

   需要準備的材料（試劑盒不提供）：

   平頭鑷子；一次性刀片；50 ml離心管；1.5 ml離心管；水浴鍋

一、原生質(zhì)體分離：

即用型酶溶液配制

注：溶液I容易被微生物污染，使用時(shí)須嚴格按照無(wú)菌操作規范進(jìn)行或者適當分裝后使用。

55℃處理能滅活核酸酶和蛋白酶并促進(jìn)酶的溶解；

即用型酶溶液現用現配，不建議配制后凍存后再使用；

溶解好的正常酶溶液應為澄清棕黃色溶液，如使用純度不好的酶，溶液為不溶解的乳白色懸濁液，不能使用。

1. 酶解：

取生長(cháng)狀態(tài)良好的葉片切成0.5-1 mm的小條，按照0.1 克葉片（擬南芥約15-20個(gè)葉片）加5 ml即用型酶溶液的比例迅速將切割好的葉片小條浸泡于酶溶液中，避光，無(wú)需震蕩，常溫（20-25℃）酶解3小時(shí)，間歇混勻。

注：酶解時(shí)間與葉片種類(lèi)，葉片生長(cháng)狀態(tài)有關(guān)，請根據實(shí)驗需要適當調整酶解時(shí)間。3小時(shí)為擬南芥葉片推薦的酶解時(shí)間。如條件允許，可以使用微型真空泵常溫避光條件下抽真空30 min，以使酶溶液更好地進(jìn)入細胞間隙。酶溶液變?yōu)榫G色表明有原生質(zhì)體已經(jīng)有分離，溶液為濃綠色表明原生質(zhì)體已經(jīng)大量分離。擬南芥原生質(zhì)體大小約為30-50 μm，顯微鏡鏡檢后確定是否分離。葉片原生質(zhì)體細胞顯微鏡下為綠色圓球狀，葉綠體分散在整個(gè)細胞內，說(shuō)明狀態(tài)較好；如呈現不規則形狀，說(shuō)明原生質(zhì)

體破碎或即將破碎。

 2. 漂洗：

        取適量溶液II-漂洗溶液（5×），用超純水稀釋為1×漂洗溶液。

酶解后加入等體積的1×漂洗溶液，如使用5 ml酶解體系，加入5 ml 1×漂洗溶液，輕柔混勻。

3. 過(guò)濾：

用孔徑70 μm篩網(wǎng)過(guò)濾步驟2中的溶液，去除未消化的葉片，用5-10 ml 1×漂洗溶液沖洗酶解器皿和未消化的葉片1-2次，將所有的液體收到50 ml離心管中。

4. 第一次收集：

濾出液100g常溫離心 2分鐘，盡量去除上清。

注：為了避免原生質(zhì)體離心時(shí)貼在管壁，建議整個(gè)實(shí)驗過(guò)程使用水平轉頭；離心時(shí)，可調低離心機的升速和降速。升速過(guò)快，原生質(zhì)體可能離到管壁上；降速過(guò)快，可能導致管底原生質(zhì)體懸起。建議升速和降速分別都使用3。

5. 第二次收集：

加入2-5 ml 1×漂洗溶液，用剪尖的藍吸頭重懸原生質(zhì)體，冰浴30分鐘，原生質(zhì)體在重力

作用下可以沉降到離心管底部，盡量吸除上清，收集原生質(zhì)體。(如果發(fā)現原生質(zhì)體沉降的速度比較慢或者得率比較低，也可以考慮常溫100 g離心1-2 min收集原生質(zhì)體)。

6. 重懸：

小心去除上清溶液，不要觸動(dòng)原生質(zhì)體沉淀，沉淀用剪尖的藍吸頭重懸于1 ml溶液III中即為原生質(zhì)體溶液。（可以用血球計數板計數，根據原生質(zhì)體數量調整溶液III的加入體積，使得原生質(zhì)體密度為2×10⁵/ml或更高）。

注：制備好的原生質(zhì)體可以在4oC或冰浴保存至少24 h。

二、實(shí)驗示例：

                使用植物原生質(zhì)體制備試劑盒轉染擬南芥原生質(zhì)體的效果圖。

實(shí)驗步驟：稱(chēng)取0.48 g轉化試劑于2 ml 離心管中，加入轉化試劑溶解液后，顛倒混勻，蒸餾水定容至2 ml，使轉化試劑充分溶解后備用。在2 ml的圓底離心管中加入10 μl（20 μg）EGFP質(zhì)粒 (植物用綠色熒光蛋白)，加入100 μl制備好的原生質(zhì)體，輕柔混勻后加入110 μl當日配制好的轉化試劑溶液，輕柔混勻，常溫靜置5 min后加入440 μl 1×漂洗溶液終止轉化，輕輕顛倒混勻，常溫100 g離心1 min，去除上清，再加入0.5 ml 1×漂洗溶液，輕柔重懸原生質(zhì)體，常溫100 g離心1 min后，盡量去除上清，收集原生質(zhì)體。加入1 ml溶液V，小心重懸原生質(zhì)體后水平放置25℃培養過(guò)夜(約16h)，次日于熒光顯微鏡下檢測EGFP熒光信號。

植物原生質(zhì)體提取試劑盒發(fā)表文章列表（21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.

實(shí)驗植物：小麥

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. 

實(shí)驗植物：煙草

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

實(shí)驗植物：煙草

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

實(shí)驗植物：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

實(shí)驗植物：煙草

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

實(shí)驗植物：擬南芥

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

實(shí)驗材料：雨生紅球藻 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

實(shí)驗材料：睡蓮 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

實(shí)驗材料：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

實(shí)驗材料：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

實(shí)驗材料：擬南芥

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

實(shí)驗材料：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

實(shí)驗材料：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.

實(shí)驗材料：小麥

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.

實(shí)驗材料：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.

實(shí)驗材料：擬南芥

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.

實(shí)驗材料：煙草

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.

實(shí)驗材料：煙草

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

實(shí)驗材料：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.

實(shí)驗材料：小麥

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.

實(shí)驗材料：煙草

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