植物原生質(zhì)體轉(zhuǎn)化試劑盒                                      

●  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(xì)胞壁由質(zhì)膜包被的具有生命活力的裸露細(xì)胞。它具有細(xì)胞生命特征和全能型，是細(xì)胞無性系變異和突變體篩選的重要來源，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。植物原生質(zhì)體轉(zhuǎn)化試劑盒(Plant Protoplasts Transformation Kit)采用的是聚乙二醇轉(zhuǎn)化方法，借助聚乙二醇短時(shí)間內(nèi)對(duì)原生質(zhì)體產(chǎn)生的沖擊效應(yīng)，使質(zhì)粒DNA、RNA或蛋白得以進(jìn)入原生質(zhì)體，經(jīng)轉(zhuǎn)化的植物原生質(zhì)體經(jīng)過一定時(shí)間培養(yǎng)后，可用于檢測(cè)外源基因的表達(dá)和功能，轉(zhuǎn)化后基因敲除或基因編輯效果等。

對(duì)于擬南芥原生質(zhì)體，使用本試劑盒轉(zhuǎn)化質(zhì)粒后通常2-6小時(shí)可以檢測(cè)相關(guān)基因的轉(zhuǎn)錄變化(mRNA等RNA的變化)，通常2-16小時(shí)后可以檢測(cè)到相關(guān)蛋白表達(dá)的變化，通常24小時(shí)可以檢測(cè)到基因編輯的效果。

本試劑盒可以用于相當(dāng)于6孔板40個(gè)孔的樣品，12孔板20個(gè)孔的樣品，或24孔板40個(gè)孔的樣品的轉(zhuǎn)化。

本試劑盒不含有原生質(zhì)體制備試劑，需要制備原生質(zhì)體請(qǐng)參見植物原生質(zhì)體制備試劑盒（RTU4082 ）。

● 貯存和效期：

   按照溫度貯存，有效期一年。轉(zhuǎn)化試劑現(xiàn)用現(xiàn)配。

試劑盒常溫運(yùn)輸。

● 使用說明：

   需要準(zhǔn)備的材料（試劑盒不提供）：

   平頭鑷子；一次性刀片；50ml離心管；1.5ml離心管；水浴鍋

 一、原生質(zhì)體轉(zhuǎn)化步驟：

1. 轉(zhuǎn)化溶液配制：

植物原生質(zhì)體轉(zhuǎn)化參考下表根據(jù)樣品量配制轉(zhuǎn)化溶液。

轉(zhuǎn)化溶液現(xiàn)用現(xiàn)配。轉(zhuǎn)化溶液需要在轉(zhuǎn)化前至少1小時(shí)配制，以確保轉(zhuǎn)化試劑溶解充分。轉(zhuǎn)化溶液配制后盡量當(dāng)天使用。配制好的轉(zhuǎn)化溶液4℃保存3-5天之內(nèi)仍然有較好的轉(zhuǎn)化效率，但和當(dāng)天配制的轉(zhuǎn)化溶液相比，轉(zhuǎn)化效果可能會(huì)有一定程度的下降。

2. 準(zhǔn)備質(zhì)粒：

取10 μl質(zhì)粒DNA(10-20 μg，濃度為1-2 μg/μl)于1.5 ml 離心管中。

注：質(zhì)粒大小建議為5-10 kb，質(zhì)粒濃度為1-2 μg/μl左右；原生質(zhì)體轉(zhuǎn)化對(duì)于質(zhì)粒的純度要求較高，盡量使用高純度的質(zhì)粒。多個(gè)質(zhì)粒同時(shí)轉(zhuǎn)化時(shí)的體積和總質(zhì)量保持不變。多個(gè)質(zhì)粒轉(zhuǎn)化時(shí)，每種質(zhì)粒的用量比例，需要酌情自行調(diào)整。質(zhì)粒轉(zhuǎn)化也可以在12或24孔板中進(jìn)行，轉(zhuǎn)化體系也可以按照比例放大或縮小。本試劑盒中描述的用量相當(dāng)于是6孔板中的用量。原生質(zhì)體用多孔板培養(yǎng)時(shí)，最好選擇未經(jīng)表面處理的多孔板。對(duì)于經(jīng)過表面處理的動(dòng)物細(xì)胞培養(yǎng)用多孔板，可以使用5%胎牛血清或小牛血清處理孔表面數(shù)秒，這樣有助于減小孔底表面對(duì)于原生質(zhì)體的損傷。

3. 質(zhì)粒轉(zhuǎn)化：

3.1 質(zhì)粒管中加入100 μl原生質(zhì)體溶液（原生質(zhì)體密度為2×10⁵/ml，約2萬個(gè)原生質(zhì)體），輕柔混勻。

3.2 加入等體積即110 μl 步驟1事先準(zhǔn)備好的轉(zhuǎn)化溶液，輕彈管底，輕柔混勻，常溫25℃放置5-15分鐘。

注：最長(zhǎng)可以孵育15 min，但通常孵育5min時(shí)間已經(jīng)足夠。最佳的孵育時(shí)間對(duì)于不同的原生質(zhì)體和不同的質(zhì)粒需要通過實(shí)驗(yàn)摸索。

4. 終止轉(zhuǎn)化：

加入2倍體積轉(zhuǎn)化終止溶液即440 μl，輕柔徹底混勻，終止轉(zhuǎn)化過程。

5. 收集原生質(zhì)體：

常溫100g離心1-2分鐘，盡量去除上清。

注：由于轉(zhuǎn)化后的溶液會(huì)非常粘稠，加入轉(zhuǎn)化終止溶液后離心1 min通?？梢允乖|(zhì)體聚集在管底，但使用某些突變體時(shí)為減少原生質(zhì)體損失，可將離心時(shí)間延長(zhǎng)到2 min。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(yàn)過程使用水平轉(zhuǎn)頭；離心時(shí)，可調(diào)低離心機(jī)的升速和降速。升速過快，原生質(zhì)體可能離到管壁上；降速過快，可能導(dǎo)致管底原生質(zhì)體懸起。建議升速和降速分別都使用3。

6. 原生質(zhì)體漂洗：

  加入500 μl 轉(zhuǎn)化終止溶液輕輕重懸原生質(zhì)體，常溫100g離心1 min，盡量去除殘留的上清。

注：本步驟可以充分去除殘留的轉(zhuǎn)化試劑溶液中的組分，避免轉(zhuǎn)化試劑溶液中的組分對(duì)于后續(xù)的不良影響。

7. 原生質(zhì)體重懸：

沉淀中加入1 ml培養(yǎng)溶液，輕柔重懸。

8. 原生質(zhì)體培養(yǎng)：

將離心管水平放置，23-25oC弱光培養(yǎng)。

注：根據(jù)實(shí)驗(yàn)需求確定孵育時(shí)間。RNA分析孵育2-6小時(shí)；酶活性分析和蛋白標(biāo)記實(shí)驗(yàn)孵育2-16小時(shí)；基因編輯的效果在轉(zhuǎn)化24小時(shí)后可能被檢測(cè)到。

                                   使用植物原生質(zhì)體轉(zhuǎn)化試劑盒轉(zhuǎn)化擬南芥原生質(zhì)體的效果圖。

實(shí)驗(yàn)步驟：稱取0.48 g轉(zhuǎn)化試劑于2 ml 離心管中，加入轉(zhuǎn)化試劑溶解液后，顛倒混勻，蒸餾水定容至2 ml，使轉(zhuǎn)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(dāng)日配制好的轉(zhuǎn)化試劑溶液，輕柔混勻，常溫靜置5 min后加入440 μl轉(zhuǎn)化終止溶液終止轉(zhuǎn)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 轉(zhuǎn)化終止溶液，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培養(yǎng)溶液，小心重懸原生質(zhì)體后水平放置25℃培養(yǎng)過夜(約16h)，次日于熒光顯微鏡下檢測(cè)EGFP熒光信號(hào)。

植物原生質(zhì)體提取試劑盒發(fā)表文章列表（19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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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(yàn)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