植物生理学报 Plant Physiology Journal 2014, 50 (3): 351~357351
收稿 2013-11-21 修定 2014-01-12
资助
国家重点基础研究发展计划(2012CB944803)、浙江省青年科学基金(LQ13C020002)和浙江省博士后项目择优资助(Bsh1202082和Bsh1202081)。
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通讯作者(E-mail: yzhu1974@; Tel: 0571-********)。
一种快速高效的水稻原生质体制备和转化方法的建立
段炼1,2, 钱君2, 郭小雨2, 朱英2,*
1
浙江师范大学化学与生命科学学院, 浙江金华321004; 2浙江省农业科学院病毒学与生物技术研究所, 浙江省植物有害生物防控重点实验室, 省部共建国家重点实验室培育基地, 杭州310021
摘要: 在模式植物拟南芥中, 原生质体瞬时表达技术已被广泛地应用到功能基因组学的研究中, 但水稻原生质体因其制备过程相对繁琐, 转化效率偏低, 尚未在基因功能研究中获得广泛应用。本研究在拟南芥原生质体制备和转化的基础之上, 对水稻原生质体的制备和转化方法进行改良优化。以水稻幼茎为起始材料, 采用纤维素酶R-10和果胶酶R-10, 对水稻组织进行消化并利用蔗糖密度梯度自沉降的方法分离原生质体, 获得了高纯度的原生质体。对质粒转化原生质体时的转化方法、转化时间及质粒浓度进行探索, 在缩短原生质体分离时间的同时, 大大提高了转化效率。用较少量的质粒DNA 即可获得外源基因在原生质体内高效的表达, 且转化效率可达70%。我们建立的这种快速有效的水稻原生质体制备和转化方法, 可为水稻功能基因组学研究提供技术支持。关键词: 水稻; 原生质体; 自沉降法
A Rapid and Efficient Method for Isolation and Transformation of Rice Protoplast
DUAN Lian 1,2, QIAN Jun 2, GUO Xiao-Yu 2, ZHU Ying 2,*1
College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, Zhejiang 321004, China; 2State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Abstract: Transient gene expression in protoplast is a common approach for studying subcelluar localization, promoter activities and protein complexes in model plant, Arabidopsis. However, protoplast isolation, transformation and as well as downstream analyses in rice are often hampered by a number of factors such as time-consuming, cost-intensive and low transformation efficiency. In this study, we reported a rapid and efficient method for isolation and transformation of rice protoplast, based on the recently published procedure for Arabidopsis protoplast preparation. 10-to-14-days stem tissues but not leaf tissues were digested by proper cellulase R-10 and mecerozyme R-10. Pure protoplasts without cell debris were isolated from the interphase of sucrose gradient after natural sink. This method was also very time-efficient, large amount of protoplasts could be obtained within one day. The transformation efficiency was pretty high (70%) with little amount of DNA. The method we presented here should be valuable for the functional genomics research in rice.Key words: rice (Oryza sativa ); protoplast; the self-settlement method
植物原生质体(protoplast)是指通过质壁分离能够分开的那部分细胞物质。离体的原生质体在适当的条件下具有繁殖、分化、再生成完整植株的能力(张红梅和王俊丽2002)。近年来, 原生质体瞬时表达技术被广泛应用于基因功能研究, 其原因除了原生质体易于制备外, 还与其本身可以摄取外源细胞器、病毒、质粒以及DNA 等各种大分子物质有关(An 等2005)。在双子叶植物中, 重组质粒转化原生质体的相关技术已经十分纯熟, PEG 法介导的原生质体转化是实现该技术的重要手段之一。早在1989年, 就有Damm 等(1989)成功将外源基因转入了模式植物拟南芥的原生质体中。此后,
关于拟南芥的原生质体的相关报道层出不穷。拟南芥原生质体除了被用于胞内运输机制(Chen 和Halkier 2000; Wolfenstette 等2012)的探索、蛋白的亚细胞定位、蛋白-蛋白互作、DNA 与蛋白质的相互反应(Faraco 等2011; Yoo 等2007)外, 还被用作反向遗传学研究的受体(Zhai 等2009)。
