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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。
OBJECTIVE: In the process of compartmentalization of cadmium by hyperaccumulator Sedum alfredii, mesophyll cells and other parenchyma cells containing large vacuoles play an important role. This paper aims to establish and optimize the extraction and purification of mesophyll protoplasts and vacuoles, and lay a foundation for the study on the mechanism of Cadmium sequestration in Sedum alfredii from a technical perspective, which is helpful to further understand the physiological and biochemical characteristics of cadmium hyperaccumulation Molecular mechanism. Innovative point: Optimized the extraction and purification technology of protoplast of Sedum alfredii Hance, and established the vacuole extraction method with high efficiency, good membrane integrity, large quantity and high purity. Methods: Including protoplast extraction, vacuolar crude extract and vacuolar purification. Protoplast Extraction: Take Sedum alfredii leaf, cut into 1 ~ 2 mm thin strips immersed in the preheated cell lysate, shock 2 h after filtration, centrifuged to obtain protoplasts. Bubble roughing: The protoplasts were lysed with protoplast lysate of 1-propanesulfonic acid at a concentration of 0.675 mmol / L, centrifuged to obtain a crude vacuole, and a solution of mannitol containing 0.8 mol / L mannitol was added. Bubble purification: To the initial vacuolar suspension was added to the lower volume concentration of Ficoll solution of 0.10 g / ml, density gradient centrifugation, to obtain purified vacuoles. CONCLUSION: Pretreatment of cell lysate can accelerate the cell wall degradation. Setting the lysis time to 2 h is favorable for the efficient extraction of protoplasts. It can be effective by the improvement of protoplast lysate concentration, cell protection solution concentration and gradient centrifugation and other parameters Extract vacuoles in the protoplasts of leaf cells.