Jones H, Ooms G, Jones M G
AFRC Institute of Arable Crops Research, Biochemistry Department, Harpenden, Herts, U.K.
Plant Mol Biol. 1989 Nov;13(5):503-11. doi: 10.1007/BF00027310.
Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.
电穿孔法用于评估对马铃薯原生质体瞬时基因表达很重要的参数。原生质体取自野生马铃薯短柄茄的叶片，以及栽培马铃薯品种德西蕾的叶片、块茎和悬浮细胞。在花椰菜花叶病毒（CaMV）35S启动子控制下的报告酶活性，即氯霉素乙酰转移酶（CAT），取决于用于电穿孔的场强和脉冲持续时间。使用持续时间为85毫秒的场脉冲，获得最大CAT活性的最佳场强为：短柄茄叶肉原生质体——250伏/厘米；德西蕾叶肉原生质体——225伏/厘米；德西蕾悬浮培养原生质体——225伏/厘米；德西蕾块茎原生质体——150伏/厘米。最佳场强与电穿孔的原生质体大小呈负相关；这与生物物理理论一致。在时间进程中，（德西蕾叶肉原生质体中的）最大CAT活性在电穿孔后36 - 48小时出现。在优化条件下对由II类马铃薯块茎蛋白启动子与β-葡萄糖醛酸酶（gus）基因连接而成的嵌合基因进行检测，结果表明，在块茎和叶肉原生质体中（DNA浓度在0 - 10皮摩尔/毫升之间），其表达与CaMV 35S启动子相当。在较高DNA浓度（20 - 30皮摩尔/毫升）下，马铃薯块茎蛋白启动子指导的gus表达水平高出4 - 5倍。文中讨论了该研究对于基因表达研究，特别是马铃薯同源基因研究的意义和潜在贡献。
