Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).
Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).
复制标题
高效叶肉原生质体分离和 PEG 介导的瞬时基因表达，用于木薯 (Manihot esculenta Crantz) 的快速和大规模基因表征
DOI：
10.1186/s12896-017-0349-2
复制
发表时间：
2017-03-14
影响因子：
3.5
通讯作者：
Liu JP
中科院分区：
文献类型：
--
作者：
Wu JZ;Liu Q;Geng XS;Li KM;Luo LJ;Liu JP
木薯（Manihot esculentaCrantz）是热带地区广泛种植的主要作物，既是重要的热量来源，也是生物燃料生产的有希望的来源。虽然稳定的基因表达已被用于转基因育种和基因功能研究，但快速、简便、大规模的转化平台已成为基因功能鉴定的迫切需要，方法以木薯试管苗完全展开的叶片为材料，对纤维素酶R-10和macerozyme R-10的浓度进行优化，10，以获得具有最高产量和活力的原生质体。确定了木薯原生质体瞬时表达的最佳条件（PEG 4000浓度和转染时间）。此外，所建立的协议的可靠性被证实为亚细胞蛋白localization.ResultsIn这项工作中，我们优化了主要影响因素，并开发了一个有效的叶肉原生质体分离和PEG介导的瞬时基因表达木薯。确定了适宜的酶解体系：1.6%纤维素酶R-10和0.8%破碎酶R-10在25 °C、黑暗条件下酶解16 h，叶肉原生质体产量和活力分别为4.4 × 107个/g FW和92.6%。当原生质体/载体DNA混合物与25%PEG 4000共孵育10 min时，获得最大的转染效率（70.8%）。我们验证了该系统的适用性，为研究亚细胞定位的MeSTP 7（一个H+/单糖共转运蛋白）与我们的瞬时表达协议和heterogouscystopsistensient基因expression system.ConclusionWe优化的主要影响因素，并开发了一个有效的叶肉原生质体分离和瞬时表达基因在木薯，这将有利于大规模表征基因和途径在木薯。
BackgroundCassava (Manihot esculentaCrantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced.MethodsFully expanded leaves from in vitro plantlets ofManihot esculentawere used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization.ResultsIn this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologousArabidopsistransient gene expression system.ConclusionWe optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.