东南景天叶肉细胞原生质体和液泡的分离与纯化技术（英文）

发布时间：2018-07-05 04:00

本文选题：超积累植物 + 东南景天　；参考：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2017年01期

【摘要】：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。
[Abstract]:Aim: to study the role of mesophyll cells and other parenchyma cells containing large vacuoles in the process of cadmium differentiation. The purpose of this paper is to establish and optimize the extraction and purification of protoplasts and vacuoles from mesophyll cells in order to lay a foundation for the study of the mechanism of cadmium segregation in Sedum southeastern China on a technical level, and to further study the physiological and molecular mechanism of cadmium superaccumulation. Innovation: the extraction and purification technology of protoplast from the leaves of Sedum southeastern China was optimized, and a vacuolar extraction method with good membrane integrity, high quantity and high purity was established. Methods: protoplast extraction, vacuolar extraction and vacuolar purification were included. The protoplasts were extracted from the leaves of Sedum davidiana. The leaves were cut into 1 ~ 2 mm thin strip and immersed in the preheated cell lytic solution. The protoplasts were filtered after 2 hours of shock and then centrifuged and washed to obtain the protoplasts. Crude vacuolar extraction: the protoplast with 0.675 mmol / L of 1-propanesulfonic acid concentration was used to split the protoplast. After centrifugation, the crude vacuole was obtained, and the vacuolar protection solution containing 0.8 mol / L mannitol was added. Vacuolar purification: Ficoll solution with mass ratio of 0. 10 g/ml was added to the lower layer of the suspension to obtain the purified vacuole by density gradient centrifugation. Conclusion: the cell wall degradation can be accelerated by preheating the cell lysate, and the cleavage time of 2 h is favorable to the efficient extraction of the protoplast, and the parameters such as the concentration of the protoplast lysate, the concentration of the cell protective liquid and the gradient centrifugation are improved. Vacuoles in protoplasts of leaf cells can be extracted effectively.
【作者单位】： MOE
【基金】：Project supported by the National Natural Science Foundation of China(Nos.31370040 and 41401366) the Zhejiang Provincial Natural Science Foundation of China(No.LR14C150001)
【分类号】：S682.33;X835

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