植物原生(shēng)質體(tǐ)制備試劑盒
貨号
|
名稱
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包裝
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RTU4082
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植物原生(shēng)質體(tǐ)制備試劑盒
|
5 ml×20次
|
● 産品組成:
序号
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組分(fēn)貨号
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名稱
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規格
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貯存
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運輸
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1
|
RTU4082-01
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溶液I-酶溶解溶液(2×)
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50
ml
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-20℃
|
RT
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2
|
RTU4082-02
|
溶液II-漂洗溶液(5×)
|
50
ml
|
-20℃
|
RT
|
3
|
RTU4082-03
|
溶液III-重懸溶液
|
25
ml
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-20℃
|
RT
|
4
|
BME
|
還(hái)原劑
|
1
ml
|
RT
|
RT
|
5
|
BSA-02
|
50mg/ml
BSA溶液
|
5
ml
|
-20℃
|
RT
|
6
|
CYL0014
|
Enzyme
Premix酶混合物
|
2
g
|
-20℃
|
RT
|
7
|
RT-1070
|
70
μm細胞過濾器
|
5個/包
|
RT
|
RT
|
|
|
說(shuō)明書(shū)
|
一份
|
|
|
● 産品簡介:
植物原生(shēng)質體(tǐ)是指脫去(qù)全部細胞壁由質膜包被的具有生(shēng)命活力的裸露細胞。它具有細胞生(shēng)命特征和全能型,是細胞無性系變異和突變體(tǐ)篩選的重要來(lái)源,同時也是植物遺傳工(gōng)程的理(lǐ)想受體(tǐ)和遺傳改良的理(lǐ)想材料。酶解法分(fēn)離(lí)原生(shēng)質體(tǐ)是一個常用的技術(shù),其原理(lǐ)是植物細胞壁主要由纖維素、半纖維素和果膠質組成,因而使用纖維素酶、半纖維素酶、離(lí)析酶和果膠酶能降解細胞壁成分(fēn),去(qù)除細胞壁,即可(kě)得(de)到原生(shēng)質體(tǐ)。試劑盒配套的酶混合物是纖維素酶R-10和離(lí)析酶R-10的混合物,優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(shēng)質體(tǐ)。
按照(zhào)每次使用5
ml酶消化體(tǐ)系計(jì)算,本試劑盒可(kě)用于20次酶消化反應。本試劑盒每個5 ml反應體(tǐ)系可(kě)處理(lǐ)0.1 g左右的拟南(nán)芥葉片(約10~15葉片),操作(zuò)較好的情況下每5 ml體(tǐ)系可(kě)獲得(de)約50-70萬個原生(shēng)質體(tǐ)(不同植物不同操作(zuò)會有一定的差異),可(kě)滿足約25-70個樣品的原生(shēng)質體(tǐ)質粒轉染操作(zuò)(按照(zhào)每樣1-2萬個細胞計(jì)算)。
本試劑盒不含有原生(shēng)質體(tǐ)轉化試劑,需要轉化請(qǐng)參見(jiàn)植物原生(shēng)質體(tǐ)轉化試劑盒(RTU4092 )。
● 貯存、效期及運輸:
按照(zhào)标簽溫度保存,至少一年(nián)有效。-20℃貯存組分(fēn)4℃存放(fàng)3-4不會影(yǐng)響使用效果。3-4天經常使用的情況,可(kě)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(lí)心管;1.5 ml離(lí)心管;水浴鍋
一、原生(shēng)質體(tǐ)分(fēn)離(lí):
即用型酶溶液配制
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5 ml配制量
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溶液I(2×)
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2.5
ml
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酶混合物
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0.095克
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55℃處理(lǐ)10分(fēn)鍾,間歇混勻,冷(lěng)卻到常溫後加入以下試劑
|
還(hái)原劑
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2.5
μl
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50mg/ml
BSA
|
0.1
ml
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滅菌水
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定容至5 ml
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注:溶液I容易被微生(shēng)物污染,使用時須嚴格按照(zhào)無菌操作(zuò)規範進行或者适當分(fēn)裝後使用。
55℃處理(lǐ)能滅活核酸酶和蛋白(bái)酶并促進酶的溶解;
即用型酶溶液現用現配,不建議(yì)配制後凍存後再使用;
溶解好的正常酶溶液應爲澄清棕黃(huáng)色溶液,如(rú)使用純度不好的酶,溶液爲不溶解的乳白(bái)色懸濁液,不能使用。
1. 酶解:
取生(shēng)長狀态良好的葉片切成0.5-1 mm的小條,按照(zhào)0.1
克葉片(拟南(nán)芥約15-20個葉片)加5
ml即用型酶溶液的比例迅速将切割好的葉片小條浸泡于酶溶液中,避光(guāng),無需震蕩,常溫(20-25℃)酶解3小時,間歇混勻。
注:酶解時間與葉片種類,葉片生(shēng)長狀态有關,請(qǐng)根據實驗需要适當調整酶解時間。3小時爲拟南(nán)芥葉片推薦的酶解時間。如(rú)條件(jiàn)允許,可(kě)以使用微型真空泵常溫避光(guāng)條件(jiàn)下抽真空30 min,以使酶溶液更好地進入細胞間隙。酶溶液變爲綠色表明有原生(shēng)質體(tǐ)已經有分(fēn)離(lí),溶液爲濃綠色表明原生(shēng)質體(tǐ)已經大(dà)量分(fēn)離(lí)。拟南(nán)芥原生(shēng)質體(tǐ)大(dà)小約爲30-50 μm,顯微鏡鏡檢後确定是否分(fēn)離(lí)。葉片原生(shēng)質體(tǐ)細胞顯微鏡下爲綠色圓球狀,葉綠體(tǐ)分(fēn)散在整個細胞内,說(shuō)明狀态較好;如(rú)呈現不規則形狀,說(shuō)明原生(shēng)質
體(tǐ)破碎或即将破碎。
2. 漂洗:
取适量溶液II-漂洗溶液(5×),用超純水稀釋爲1×漂洗溶液。
|
1×漂洗溶液
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組份
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50
ml配制量
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溶液II-漂洗溶液(5×)
|
10
ml
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滅菌水
|
40
ml
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酶解後加入等體(tǐ)積的1×漂洗溶液,如(rú)使用5
ml酶解體(tǐ)系,加入5 ml 1×漂洗溶液,輕柔混勻。
3. 過濾:
用孔徑70 μm篩網過濾步驟2中的溶液,去(qù)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,将所有的液體(tǐ)收到50
ml離(lí)心管中。
4. 第一次收集:
濾出液100g常溫離(lí)心 2分(fēn)鍾,盡量去(qù)除上清。
注:爲了避免原生(shēng)質體(tǐ)離(lí)心時貼在管壁,建議(yì)整個實驗過程使用水平轉頭;離(lí)心時,可(kě)調低離(lí)心機(jī)的升速和降速。升速過快(kuài),原生(shēng)質體(tǐ)可(kě)能離(lí)到管壁上;降速過快(kuài),可(kě)能導緻管底原生(shēng)質體(tǐ)懸起。建議(yì)升速和降速分(fēn)别都(dōu)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(shēng)質體(tǐ),冰浴30分(fēn)鍾,原生(shēng)質體(tǐ)在重力
作(zuò)用下可(kě)以沉降到離(lí)心管底部,盡量吸除上清,收集原生(shēng)質體(tǐ)。(如(rú)果發現原生(shēng)質體(tǐ)沉降的速度比較慢(màn)或者得(de)率比較低,也可(kě)以考慮常溫100 g離(lí)心1-2
min收集原生(shēng)質體(tǐ))。
6. 重懸:
小心去(qù)除上清溶液,不要觸動原生(shēng)質體(tǐ)沉澱,沉澱用剪尖的藍吸頭重懸于1 ml溶液III中即爲原生(shēng)質體(tǐ)溶液。(可(kě)以用血球計(jì)數闆計(jì)數,根據原生(shēng)質體(tǐ)數量調整溶液III的加入體(tǐ)積,使得(de)原生(shēng)質體(tǐ)密度爲2×105/ml或更高)。
注:制備好的原生(shēng)質體(tǐ)可(kě)以在4oC或冰浴保存至少24 h。
二、實驗示例:
使用植物原生(shēng)質體(tǐ)制備試劑盒轉染拟南(nán)芥原生(shēng)質體(tǐ)的效果圖。
實驗步驟:稱取0.48 g轉化試劑于2 ml 離(lí)心管中,加入轉化試劑溶解液後,颠倒混勻,蒸餾水定容至2 ml,使轉化試劑充分(fēn)溶解後備用。在2
ml的圓底離(lí)心管中加入10 μl(20 μg)EGFP質粒
(植物用綠色熒光(guāng)蛋白(bái)),加入100 μl制備好的原生(shēng)質體(tǐ),輕柔混勻後加入110 μl當日(rì)配制好的轉化試劑溶液,輕柔混勻,常溫靜(jìng)置5
min後加入440 μl 1×漂洗溶液終止轉化,輕輕颠倒混勻,常溫100 g離(lí)心1
min,去(qù)除上清,再加入0.5 ml 1×漂洗溶液,輕柔重懸原生(shēng)質體(tǐ),常溫100
g離(lí)心1 min後,盡量去(qù)除上清,收集原生(shēng)質體(tǐ)。加入1 ml溶液V,小心重懸原生(shēng)質體(tǐ)後水平放(fàng)置25℃培養過夜(約16h),次日(rì)于熒光(guāng)顯微鏡下檢測EGFP熒光(guāng)信号。
植物原生(shēng)質體(tǐ)提取試劑盒發表文章(zhāng)列表
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
實驗植物:拟南(nán)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
實驗材料:雨(yǔ)生(shēng)紅(hóng)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
實驗材料:拟南(nán)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