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。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。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。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 is to investigate the possibility of zinc (Zn) biofortification in the grains of rice (Oryza sativa L.) by inoculation of endophytic strains isolated from a Zn hyperaccumulator, Sedum alfredii Hance. Five endophytic strains, Burkholderia sp. SaZR4, Burkholderia sp. SaMR10, Sphingomonas sp. SaMR12, Variovorax sp. SaNR1, and Enterobacter sp. SaCS20, isolated from S. alfredii, were inoculated in the roots of Japonica rice Nipponbare under hydroponic condition. Fluorescence images showed that endophytic strains successfully colonized rice roots after 72 h. Improved root morphology and plant growth of rice was observed after inoculation with endophytic strains especially SaMR12 and SaCS20. Under hydroponic conditions, endophytic inoculation with SaMR12 and SaCS20 increased Zn concentration by 44.4% and 51.1% in shoots, and by 73.6% and 83.4% in roots, respectively. Under soil conditions, endophytic inoculation with SaMR12 and SaCS20 resulted in an increase of grain yields and elevated Zn concentrations by 20.3% and 21.9% in brown rice and by 13.7% and 11.2% in polished rice, respectively. After inoculation of SaMR12 and SaCS20, rhizosphere soils of rice plants contained higher concentration of DTPA-Zn by 10.4% and 20.6%, respectively. In situ micro-X-ray fluorescence mapping of Zn confirmed the elevated Zn content in the rhizosphere zone of rice treated with SaMR12 as compared with the control. The above results suggested that endophytic microbes isolated from S. alfredii could successfully colonize rice roots, resulting in improved root morphology and plant growth, increased Zn bioavailability in rhizosphere soils, and elevated grain yields and Zn densities in grains.