| 摘要: |
| 本研究旨在构建高州油茶（Camellia drupifera）叶肉原生质体的高效分离与瞬时转化技术体系。以高州油茶五月龄实生苗真叶为实验材料，采用正交试验系统优化纤维素酶、果胶酶与离析酶的协同作用浓度配比，以及渗透压调节剂和酶解时间等关键解离参数，并构建基于聚乙二醇（PEG-4000）介导的原生质体瞬时转化体系。结果表明，1.0%纤维素酶、0.4%果胶酶、0.2%离析酶和0.4 mol/L甘露醇的酶解液体系，原生质体产量和细胞活力分别为5.67×105 cells/g·FW和55.78%，辅以20 min真空抽压及6 h酶解时间，产量和细胞活力显著提升（P <0.05）至6.83×105 cells/g·FW和65.70%。将携带35S强启动子的绿色荧光蛋白报告基因（p35S::eGFP）质粒与50% PEG-4000缓冲液混合转染原生质体，黑暗中孵育20 min，瞬时转化效率达74.63%。本研究成功建立了一种高效简单的高州油茶原生质体分离和瞬时转化系统，该系统对油茶研究的体细胞融合、基因组编辑、蛋白功能研究等多个领域具有重要意义。 |
| 关键词: 高州油茶 原生质体制备 叶肉细胞 纯化 瞬时转化 |
| DOI： |
| 投稿时间：2025-04-25修订日期：2025-05-30 |
| 基金项目: |
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| Development of an Efficient Mesophyll Protoplast Isolation and Transient Transformation System in Camellia drupifera |
|
Liweimeng, Liyongquan
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| (仲恺农业工程学院) |
| Abstract: |
| In this study, we aimed to establish an efficient system for isolating mesophyll protoplasts and achieving transient transformation in Camellia drupifera.Using true leaves from five-month-old seedlings, we optimized the synergistic concentration ratios of cellulase, pectinase, and macerozyme, along with key dissociation parameters (osmotic regulators and digestion time) via orthogonal experimental design, and developed a polyethylene glycol (PEG-4000)-mediated transient transformation system. Results showed that an enzymatic solution containing 1.0% cellulase, 0.4% pectinase, 0.2% macerozyme, and 0.4 mol/L mannitol yielded protoplasts at 5.67×10? cells/g·FW with 55.78% viability; supplemented with 20 min vacuum pumping and 6 h digestion, yield and viability significantly increased (P <0.05) to 6.83×10? cells/g·FW and 65.70%. Transient transformation efficiency reached 74.63% after transfecting protoplasts with a p35S::eGFP reporter plasmid (driven by the 35S promoter) using 50% PEG-4000 buffer, followed by 20 min dark incubation. This study successfully established a simple yet efficient protoplast isolation and transient transformation system for C. drupifera, which holds significant potential for somatic hybridization, genome editing, and protein functional studies in oil-tea camellia research. |
| Key words: Camellia drupifera Protoplast isolation Mesophyll cells Purifcation Transient transformation |
