| 摘要: |
| 运用电脉冲转基因仪对合浦珠母贝卵子进行电脉冲处理以优化电穿孔法转基因的电击参数。实验中调制百分比:100%;脉冲间隔:1.0s;电极距离:2mm;脉冲个数:5个,参数保持不变。用不同的电脉冲参数组合(脉冲电压100～400V、脉冲持续时间0.5～3.0ms、脉冲频率10～50kHz,共28个组合)对卵子电击,然后加入精子进行授精,统计各组受精率和孵化率,以受精率和孵化率达到50%以上为衡量指标。结果表明,合浦珠母贝卵子对低脉冲频率和高电压非常敏感,当频率低于20kHz或脉冲电压高于400V时对卵子损伤较大。脉冲持续时间对其影响不大,适应范围较宽。优化的转基因电击脉冲参数组合为:脉冲电压1.0kV/cm,脉冲持续时间2.0ms,脉冲频率50kHz,或者脉冲电压1.5kV/cm,脉冲持续时间1.5ms,脉冲频率50kHz。 |
| 关键词: 脉冲参数 合浦珠母贝 卵子 转基因 电穿孔法 |
| DOI： |
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| 基金项目:广东省科技计划项目(2002B2150101) |
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| Optimization of pulse parameters in electroporation of Pinctada fucata eggs |
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| Abstract: |
| The electroporation of Pinctada fucata eggs was carried out to determine optimal pulse parameters for gene transfer by using electroporation apparatus. The eggs of P. fucata were electroporated with 28 combinations of parameters (voltages: 100-400 V with 100 V interval, burst duration: 0.5～3.0 ms with 0.5ms interval, and radio frequency: 10～50 kHz with 10 kHz interval) while keeping the following parameters constant: 100% modulation, burst interval 1.0s and 5 bursts, and subsequently fertilized with the sperm. The rates of fertilization and hatch of each combination were calculated after 30-min fertilization. The results indicate that the eggs are sensitive to low radio frequency and high voltage. The rates of fertilization and hatch were low when radio frequency was lower than 20 kHz or voltage higher than 400 V. In contrast, the eggs could tolerate a wide range of burst duration time. These observations suggest that the optimal combination of electropation parameters are as follows: voltage 300V, burst duration 1.5msec and radio frequency 50kHz, or voltage 200V, burst duration 2.0msec and radio frequency 50kHz, under the requirements of no less than 50% rates of fertilization and hatch. |
| Key words: electroporation pulse parameter Pinctada fucata egg transgene |
