PEG介导的柳枝稷叶肉细胞原生质体瞬时表达体系的建立

Establishment of a gene transient expression system mediated by polyethylene glycol in switchgrass(Panicum virgatum)mesophyll protoplasts

为了建立以PEG介导的柳枝稷叶肉细胞原生质体瞬时表达体系,本研究以柳枝稷Alamo品种的叶片为材料,通过设计梯度实验确定柳枝稷原生质体分离的最佳条件,每组梯度实验重复3次,方案如下:1)对植物培养的时间进行优化。酶解时间固定在8h,甘露醇浓度为0.6mol/L,将培养了1,2,3和4周的黄化苗用作实验材料;2)渗透压优化。选取2周的黄化苗为实验材料,酶解时间固定在8h,甘露醇浓度分别为0.4,0.5,0.6,0.7和0.8mol/L;3)酶解时间优化。选取2周的黄化苗为实验材料,甘露醇浓度为0.6mol/L,酶解时间分别为6,7,8,9和10h。通过对比分析发现,苗龄为2周的柳枝稷黄化苗叶片在甘露醇为0.6mol/L的酶解液中,黑暗,28℃并裂解8h后分离的原生质体最为理想。接下来构建了能够在植物细胞内表达GFP-MYB103融合蛋白(GFP为绿色荧光蛋白)的重组质粒LN-OsMYB103,利用PEG介导法将质粒LN-OsMYB103转化进入柳枝稷原生质体,并且在激光共聚焦显微镜下观察到了强烈的绿色荧光蛋白信号。该结果表明,分离的原生质体可以行使正常生物学功能。综上所述,利用柳枝稷叶片建立了一套完整的柳枝稷叶肉细胞原生质体瞬时表达体系。这将为深入开展柳枝稷的功能基因组学研究奠定了基础。

Leaves of Alamo switchgrass(Panicum virgatum)were used as materials to establish a transient expression system for mesophyll protoplasts cells.To determine the optimal conditions to obtain protoplasts from switchgrass,we designed a gradient experiment,with three replicates per treatment.First,we determined the optimal age of plants as the source of material.The leaves of etiolated seedlings grown for 1,2,3,and 4weeks were subjected to enzymolysis for 8hin a solution containing D-mannitol at 0.6mol/L.Next,we determined the optimal osmotic pressure by exposing leaf material from 2-week-old etiolated seedlings to solutions contai ning D-mannitol at 0.4mol/L,0.5mol/L,0.6mol/L,0.7mol/L and 0.8mol/L.The optimal duration of enzymolysis was determined by subjecting leaf material from 2-week-old etiolated seedlings to enzymolysis for6,7,8,9,and 10 hin a solution containing 0.6mol/L D-mannitol.This series of assays showed that the optimal conditions to isolate protoplasts were as follows:2-week-old etiolated seedlings as the source material,and enzymatic hydrolysis in a solution containing 0.6 mol/L D-mannitol in the dark for 8hat 28 ℃.The protoplasts obtained using this method were transformed with the plasmid LN-OsMYB103 by apolyethylene glycolmediated method,and strong green fluorescence was detected by laser confocal microscopy.These results indicated that the protoplasts retained normal biological functions.In summary,we established an integrated transient expression system for protoplasts obtained from switchgrass leaves.This method will be very useful for functional genomics research on switchgrass.