中国沙棘子叶叶肉细胞电流全细胞记录的研究

Whole-cell Currents in the Plasma Membrane of Hippophae rhamnoides Cotyledon Mesophyll Cell Protoplasts

实验得出适合膜片钳实验的耐盐木本植物中国沙棘子叶原生质体的游离条件,即取当天脱壳的子叶,放入渗透浓度为0.88 mol/L,pH为5.8,酶液的组分为10 g/LCellulase R-10、0.5 g/LPectolyase Y-23、1.5 g/LMacrozymeR-10、1 g/L Hemicellulase、1 g/L BSA、1 g/L PVP-40、2 mmol/L CaCl2、10 mmol/L Mes和5 mmol/L Vc的溶液中,在26℃、黑暗条件下,静置4 h。并且利用Multiclamp 700B放大器和Clampex 9.2软件,记录到了该原生质体质膜全细胞内向K+通道电流(-459.137 pA及-290.527 pA)和非选择性阳离子通道的外向电流(8.036 pA)。这为沙棘属木本植物细胞的电生理特性及其耐盐性机制关系的研究奠定基础。

Protoplasts were successfully isolated from the cotyledons of Hippophae rhamnoides by incubating cotyledons which newly emerged from the seed coat in an enzyme solution at 26 ℃ in the dark for 4 h.The enzyme solution contained 10 g/L （w/v）Cellulase R-10,0.5 g/L （（w/v）Pectolyase Y- 23,1.5 g/L （ （w/v）MacrozymeR-10, 1 g/L （ （w/v）nemicellulase, 1 g/L （ （w/v）BSA, 1 g/L （ （w/v）PVP-40,2 mmol/L CaC12,10mmol/L Mes,and 5 mmol/L Vc and 0.88 mol/L D-mannitol （pH5.8）. This is the first report on the isolation of protoplasts from Hippophae rhamnoides. Moreover, the K^＋ channel currents （- 59.137 pA and -290.527 pA） and non-selective cation channel currents （8.036 pA） across the whole protoplast membrane were measnured by using a Multiclamp 700 B amplifier and the Clampex 9.2 software. The results will facilitate further investigations on the cellular electrophysiology and mechanisms of salt tolerance of woody plants in the genus of sea buckthorn （Hippophae）.