葡萄原生质体分离及瞬时转化体系的建立  

Isolation of Protoplast and Establishment of Transient Expression System in Grapevine(Vitis vinifera L.)

作　　者：舒小娟[1] 温腾建 邢佳毅[1] 卢龙[1] 胡建芳[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193

出　　处：《西北植物学报》2015年第6期1262-1268,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31471842)

摘　　要：为了建立葡萄原生质体进行遗传转化的技术,该研究以葡萄品种‘黑香蕉’的叶片和愈伤组织为材料,分析纤维素酶和离析酶的浓度与配比、渗透压和酶解时间等主要因素对葡萄原生质体分离的影响,探讨建立稳定、高效的葡萄原生质体分离与瞬时转化体系,为鉴定目标基因的功能奠定基础。结果表明:(1)葡萄叶片原生质体的分离以3.0%纤维素酶和0.75%离析酶的酶组合,在0.6mol/L甘露醇溶液中,酶解14h为宜,每克游离产量为4.09×106个原生质体,活力为83.12%。(2)葡萄愈伤组织原生质体的分离以2.0%纤维素酶和0.5%离析酶的酶组合,在0.5mol/L甘露醇溶液中,酶解14h为宜,每克游离产量为6.05×106个原生质体,活力为84.13%。(3)利用该方法得到的葡萄原生质体为受体,采用40%PEG-4000介导转化质粒载体pEZS-NL,目标基因瞬时表达产物检测表明,GFP蛋白表达稳定、清晰。该研究建立的葡萄原生质体制备和转化体系,可以用较少量的质粒DNA获得外源基因在原生质体内的表达,为葡萄功能基因的研究提供技术支持。In order to establish an efficient transient expression system based on grapevine protoplasts,we used the mesophyll and callus of grape ‘Heixiangjiao’ to analyze the key factors related to isolating effectively protoplasts,such as cellulose and macerozyme enzyme composition,concentration of mannitol in enzyme solution,duration of enzyme dissolve,and so on.The protoplast was used as a vehicle to explore the establishment of a stable,efficient grape protoplast isolation and transient transformation system,and lay the foundation for building a transient expression system.The results showed that：（1）the optimal enzyme solution for leaf protoplast isolation was 3.0% cellulase onozuka R-10＋0.75% macerozyme R-10＋0.6 mol/L mannitol.The digestion was conducted in the dark under 28 ℃ for 14 h,and the protoplasts yield was 4.09×106 per gram,the vitality was 83.12%.（2）The optimal enzyme solution for callus protoplast isolation was 2.0% cellulase onozuka R-10＋0.5% macerozyme R-10＋0.5 mol/L mannitol.The digestion was conducted in the dark under 28 ℃ for 14 h,and the protoplasts yield was 6.05×106 per gram,the vitality was 84.13%.（3）The transient expression vector pEZS-NL with reported gene coding green fluorescent protein （GFP） was transferred into protoplasts by 40% PEG-4000 method.The GFP protein expressed stably and clearly in all over the protoplast.We establish grape protoplast isolation and transformation system in this paper.The gene can be expressed efficiently in grape protoplasts with a small amount of plasmid DNA,which provides technical support for grape functional genomics studies.

关 键 词：葡萄 原生质体 叶片 愈伤组织 遗传转化 瞬时表达 

分 类 号：Q781[生物学—分子生物学]