玉米胚乳细胞原生质体的分离与流式纯化

Isolation and Flow Purification of Endosperm Protoplast from Developing Seed of Maize

玉米胚乳是籽粒淀粉形成的主要场所,胚乳细胞的发育对籽粒的器官建成具有重要意义。分离胚乳细胞原生质体,能为胚乳培养、转录组分析等后续研究提供均一实验材料。本文在借鉴前人研究的基础上,通过调整酶的搭配种类、浓度以及质膜稳定剂、渗透压稳定剂的使用,优化了一套有效的玉米籽粒胚乳原生质体分离方法,并进一步运用流式分选技术纯化原生质体。结果表明,以1%纤维素酶、0.5%离析酶、0.5%半纤维素酶,在渗透压调节剂0.7～0.8 mol L–1和质膜稳定剂0.8 mol L–1的MS培养液内,30℃消解4 h,能够获得大量完整的粗制原生质体,二乙酸荧光素(FDA)染色表明纯化的原生质体仍能保持90%以上的生活力。流式分选术可将有活力原生质体从粗制原生质体悬浮液中富集、纯化出来。对一些原生质体分离和流式分选时的技术参数和影响因素进行了讨论。

Maize endosperm is the main place for synthesizing starch and its development is very important for building grain. Isolation and purification of endosperm cell protoplasts can offer the homogeneous experimental materials for endosperm cultivation, transcriptome analysis and cell typing. On the basis of previous studies, this paper optimized a set of isolation and purification methods of maize endosperm protoplast through adjusting the combination and concentration of enzymes as well as using membrane stabilizer and osmotic stabilizer. A lot of crude protoplasts were obtained when endosperm tissue was digested in MS medium containing 0.5% macerozyme, 0.5% hemicellulase, 1.0% cellulose, 0.7–0.8 mol L-1 of osmotic stabilizer and 0.8 mol L-1 of membrane stabilizer for 4 h at 30oC. Staining of Fluorescein diacetate （FDA） showed that purified protoplasts maintained more than 90% of viability. The viable protoplasts were accumulated and purified from the crude protoplast suspension by flow sorting technology. Some technical parameters and influencing factors about protoplast isolation and flow sorting were also discussed.