本文選題：超積累植物 + 東南景天��； 參考：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2017年01期

【摘要】：目的:在超積累植物東南景天對(duì)鎘的區(qū)隔化過程中,葉肉細(xì)胞等內(nèi)含大型液泡的薄壁細(xì)胞起重要作用。本文旨在建立并優(yōu)化其葉肉細(xì)胞原生質(zhì)體和液泡的提取和純化技術(shù),在技術(shù)層面上為東南景天的鎘區(qū)隔化機(jī)理研究奠定基礎(chǔ),有助于深入探明其超積累鎘的生理與分子機(jī)理。創(chuàng)新點(diǎn):優(yōu)化了東南景天葉片原生質(zhì)體的提取和純化技術(shù),并建立了能較高效率獲得膜完整性好、數(shù)量多、純度高的液泡提取方法。方法:主要包括原生質(zhì)體提取、液泡粗提和液泡純化。原生質(zhì)體提取:取東南景天葉片,切成1~2 mm的細(xì)條狀后浸入經(jīng)預(yù)熱過的細(xì)胞裂解液中,震蕩2 h后過濾,離心清洗后獲得原生質(zhì)體。液泡粗提:采用1-丙磺酸濃度為0.675 mmol/L的原生質(zhì)體裂解液裂解原生質(zhì)體,離心后獲得粗提的液泡,并加入含0.8 mol/L甘露醇的液泡保護(hù)液。液泡純化:往初提液泡的懸浮液下層加入質(zhì)量體積比濃度為0.10 g/ml的Ficoll溶液,進(jìn)行密度梯度離心,獲取純化的液泡。結(jié)論:細(xì)胞裂解液的預(yù)熱處理可加速細(xì)胞壁降解,裂解時(shí)間設(shè)置為2 h有利于原生質(zhì)體的高效提取;通過對(duì)原生質(zhì)體裂解液濃度、細(xì)胞保護(hù)液濃度和梯度離心等參數(shù)的改良,可有效提取葉片細(xì)胞原生質(zhì)體中的液泡。
[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.
【作者單位】： 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)
【分類號(hào)】：S682.33;X835

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