拟南芥叶肉原生质体分离条件的优化研究  

Optimization Conditions of Arabidopsis Mesophyll Protoplast Isolation

作　　者：廖嘉明[1] 王伯初[1] 王益川[1] 田继权[1] 

机构地区：[1]重庆大学生物工程学院,教育部生物流变科学与技术重点实验室,重庆400044

出　　处：《西北植物学报》2010年第6期1271-1276,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(10872223)

摘　　要：以野生型拟南芥(Arabidopsis thaliana,ecotype Columbia)无菌苗为材料,研究了叶肉原生质体分离过程中的预处理条件、酶解方式、酶解温度和离心力大小等因素对产量和活力的影响。结果表明,酶解方式和酶解温度对原生质体产量影响显著,28℃静置酶解14 h能够将原生质体产量提高6.32倍。低温预处理和离心力大小对原生质体活力影响显著,4℃低温预处理24 h能够将原生质体活力提高55%。适宜拟南芥原生质体叶肉细胞分离的最佳条件为:4℃低温预处理24 h,28℃静置酶解14 h,600 r.min-1离心3次,每次10 min,得到的原生质体产量为2.91×106个.g-1,活力为84.03%。Leaves of Arabidopsis thaliana columbia plants were taken for protoplast isolation.The different factors which affected the protoplast yield and vitality,such as enzymatic dissociation methods,temperature,and centrifugal force,were investigated.The result showed that there were highly significant different among enzymatic dissociation methods,temperature in the protoplast yield,low temperature pretreatment and centrifugal force in the protoplast vitality.The yield of protoplast by incubating the leaves at 28℃ for 14 hours in enzyme solution was 6.32 times than at 25℃ for 3 hours with the method of shaking culture;low temperature pretreatment at 4℃ for 24 hours could enhance the protoplast vitality about 55%.The optimum treatment was low temperature pretreatment at 4℃ for 24 hours,incubating the leaves at 28℃ for 14 hours in enzyme solution,and centrifuging the protoplast at 600 r·min-1,the protoplast yield was 2.91×106 cells·g-1 and the vitality was 84.03%.

关 键 词：拟南芥 原生质体 游离 纯化 

分 类 号：Q503[生物学—生物化学]