玉米和拟南芥的原生质体制备及瞬时表达体系的研究

Protoplast Preparation and Transient Expression System in Maize and Arabidopsis

[目的]优化玉米和拟南芥原生质体的制备条件及瞬时表达体系.[方法]以不同时期玉米叶片和拟南芥为材料分离原生质体,通过对不同酶浓度,解离时间和渗透压的研究,优化最佳分离原生质体体系;原生质体再经PEG介导的转化,通过GFP基因表达百分比鉴定原生质体活性和转化效率.[结果]玉米和拟南芥原生质分离的最佳条件为：纤维素酶1.5％,离析酶0.5％;拟南芥酶解4h,甘露醇浓度0.4 mol/L;玉米酶解6h,甘露醇浓度0.45 ～0.50 mol/L.最佳生长时期为：拟南芥6～8片叶;玉米二片叶.用去内毒素质粒经PEG（玉米30％ PEG,拟南芥40％ PEG）诱导转化置于黑暗下培养,原生质体活性和转化效率较高,原生质体中可以观察到明显的GFP荧光.[结论]玉米和拟南芥原生质体的制备受酶浓度,解离时间和渗透压的影响,在原生质体转化过程中,质粒DNA纯度,PEG浓度等是影响转化效率的关键因素,与拟南芥原生质体相比玉米原生质体要稳定性差一些.

[ Objective ]To optimize the preparation conditions of maize and Arabidopsis protoplasts and transient expression system. [ Meth- od ] Maize and Arabidopsis leaves at different development periods were used to dissociate protoplasts, the enzyme concentrations, dissociation time and osmotic pressure were investigated and optimized using GFP expression system to identify protoplast activity and transformation effi- ciency. [ Result] The best conditions for protoplasts isolation were that 1.5% cellulase enzyme, 0.5% macerozyme and 0. 4 mol/L mannitol, digested for 4 hours for Arabidopsis ; 0. 45 - 0.50 mol/L mannitol, digested for 6 hours for maize. The best development periods were 6 - 8 leaves Arabidopsis and two-leaf corn. Endotoxin-free plasmid were used for transformation with PEG （maize 30% PEG, Arabidopsis, 40% PEG） and cultured dark. GFP fluorescence was obviously observed in protoplasts. [ Conclusion] Preparation of maize and Arabidopsis proto- plasts was affected by the enzyme concentration, the dissociation time, the leaves and osmolality. For protoplast transformation, purity of plas- mid DNA, PEG concentration was important factor for transformation efficiency. Compared with Arabidopsis, the stability of maize protoplasts was poor.