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作物学报 ›› 2014, Vol. 40 ›› Issue (03): 424-430.doi: 10.3724/SP.J.1006.2014.00424

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米胚乳细胞原生质体的分离与流式纯化

郭艳萍**,任成杰**,李志伟,王文斌,张仁和,路海东,刘建超,张兴华,薛吉全*,郭东伟*   

  1. 西北农林科技大学农学院 / 农业部西北旱区玉米生物学与遗传育种重点实验室, 陕西杨凌 712100
  • 收稿日期:2013-05-13 修回日期:2013-10-20 出版日期:2014-03-12 网络出版日期:2014-01-16
  • 通讯作者: 薛吉全,E-mail: xjq2934@163.com;郭东伟, E-mail: gdwei1973@126.com
  • 基金资助:

    本研究由西北农林科技大学基本科研业务费(Z109021123)和国家自然科学基金项目(31371626)资助。

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

GUO Yan-Ping**,REN Cheng-Jie**,LI Zhi-Wei,WANG Wen-Bin,ZHANG Ren-He,LU Hai-Dong,LIU Jian-Chao,ZHANG Xing-Hua,XUE Ji-Quan*,GUO Dong-Wei*   

  1. College of Agronomy, Northwest A&F University / Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest Region, Ministry of Agriculture, Yangling 712100, China?
  • Received:2013-05-13 Revised:2013-10-20 Published:2014-03-12 Published online:2014-01-16
  • Contact: 薛吉全,E-mail: xjq2934@163.com;郭东伟, E-mail: gdwei1973@126.com

摘要:

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0.8mol L-1MS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。

关键词: 玉米, 胚乳细胞, 原生质体, 内多倍化, 分离, 流式分选

Abstract:

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.

Key words: Maize, Endosperm, Protoplast, Endoreduplication, Isolation, Flow sorting

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