摘要: 从植物组织中释放出高质量的原生质体是建立其他技术体系如蛋白亚细胞定位、瞬时表达分析、蛋白质间互作的前提。为了快速挖掘C3-C4中间型植物松叶猪毛菜（Salsola laricifolia）的特殊功能基因，建立一个简单高效的叶片原生体的制备方法必不可少。本研究以松叶猪毛菜无菌组培苗的真叶为材料，分析不同纤维素酶和离析酶的浓度配比、渗透压对原生质体分离的影响。结果表明：采用25 d龄的无菌组培苗真叶，在2%纤维素酶+0.5%离析酶+0.6 molL-1甘露醇的酶解液中25 ℃酶解2 h，使用W5溶液在800 rpmmin-1的转速下纯化，原生质体产量可达1.21106 个，活力为85%。并且利用得到的松叶猪毛菜原生质体作为受体，用PEG 转化法成功转化pBI121-SaNADP-ME4-GFP 质粒载体，检测到SaNADP-ME4定位于叶绿体中。本项研究建立了松叶猪毛菜叶片原生质体高效制备体系，为松叶猪毛菜特殊基因功能的挖掘奠定了基础。
Abstract: The release of high- quality protoplasts from plant tissues is a prerequisite for the establishment of other technical systems such as protein subcellular localization, transient expression analysis, and protein-protein interactions. To quickly explore the special functional genes of the C3-C4 intermediate plant Salsola laricifolia, it is essential to establish a simple and efficient method for preparing leaf protoplasts. In this study, the euphylla of axenic tissue culture seedlings of S. laricifolia was used as material to analyze the effects of different concentration ratios of cellulase and isolated enzymes and osmotic pressure on protoplast isolation. The results showed that the true leaves of sterile tissue culture seedlings with a seedling age of 25 days were used for enzymatic hydrolysis in enzymatic hydrolysis solution of 2% cellulase + 0.5% isolated enzyme R-10 + 0.6 molL-1 mannitol at 25 C for 2 h, and W5 The solution was purified at a speed of 800 rpmmin-1, the yield of protoplasts could reach 1.21 106, and the viability was 85%. Using the obtained protoplast of S. laricifolia as a receptor, the pBI121-SaNADP-ME4-GFP plasmid vector was successfully transformed by the PEG transformation method, and it was detected that SaNADP- ME4 was located in the chloroplast. In this study, an efficient system for preparing protoplasts from the leaves of S. laricifolia was established, which lays the foundation for mining the special gene functions of this species.
| [V1] | 2023-05-30 18:40:00 | ChinaXiv:202305.00260V1 | 下载全文 |
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