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On-line Access: 2017-01-03

Received: 2016-03-27

Revision Accepted: 2016-09-12

Crosschecked: 2016-12-16

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Ling-li Lu

http://orcid.org/0000-0003-4754-629X

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Journal of Zhejiang University SCIENCE B 2017 Vol.18 No.1 P.85-88

http://doi.org/10.1631/jzus.B1600138


Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts


Author(s):  Xiao-yu Gao, Xing-cheng Liao, Ruo-lai Wu, Ting Liu, Hai-xing Wang, Ling-li Lu

Affiliation(s):  MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China

Corresponding email(s):   lulingli@zju.edu.cn

Key Words:  Hyperaccumulator plant, Sedum alfredii, Purified isolation, Protoplast, Vacuole


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Xiao-yu Gao, Xing-cheng Liao, Ruo-lai Wu, Ting Liu, Hai-xing Wang, Ling-li Lu. Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts[J]. Journal of Zhejiang University Science B, 2017, 18(1): 85-88.

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author="Xiao-yu Gao, Xing-cheng Liao, Ruo-lai Wu, Ting Liu, Hai-xing Wang, Ling-li Lu",
journal="Journal of Zhejiang University Science B",
volume="18",
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pages="85-88",
year="2017",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1600138"
}

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Abstract: 
This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h–long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.

东南景天叶肉细胞原生质体和液泡的分离与纯化技术

目的:在超积累植物东南景天对镉的区隔化过程中,叶肉细胞等内含大型液泡的薄壁细胞起重要作用。本文旨在建立并优化其叶肉细胞原生质体和液泡的提取和纯化技术,在技术层面上为东南景天的镉区隔化机理研究奠定基础,有助于深入探明其超积累镉的生理与分子机理。
创新点:优化了东南景天叶片原生质体的提取和纯化技术,并建立了能较高效率获得膜完整性好、数量多、纯度高的液泡提取方法。
方法:主要包括原生质体提取、液泡粗提和液泡纯化。原生质体提取:取东南景天叶片,切成1~2 mm的细条状后浸入经预热过的细胞裂解液中,震荡2 h后过滤,离心清洗后获得原生质体。液泡粗提:采用1-丙磺酸浓度为0.675 mmol/L的原生质体裂解液裂解原生质体,离心后获得粗提的液泡,并加入含0.8 mol/L甘露醇的液泡保护液。液泡纯化:往初提液泡的悬浮液下层加入质量体积比浓度为0.10 g/ml的Ficoll溶液,进行密度梯度离心,获取纯化的液泡。
结论:细胞裂解液的预热处理可加速细胞壁降解,裂解时间设置为2 h有利于原生质体的高效提取;通过对原生质体裂解液浓度、细胞保护液浓度和梯度离心等参数的改良,可有效提取叶片细胞原生质体中的液泡。

关键词:超积累植物;东南景天;原生质体;液泡;提取与纯化

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Reference

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