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）

Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts

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。

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 ℃ for 5 rain significantly accel- erated 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 vacu- ole isolation was diluted, and 0.675 mmol/L was identi- fied as the most appropriate 3-[（3-cholamidopropyl） dimethylammonio]- 1 -propanesulfonic acid （CHAPS） level, in which S. alfredii large vacuoles are charac- terized by a high metal and malic acid content.