真鲷精子在外源DNA下的生理特性和转染效果

Effect of exogenous DNA on sperm of red seabream(Pagrus major)

为研究真鲷(Pagrus major)精子在外源DNA下的生理特性和转染效果,探索真鲷精子介导转基因技术,作者通过计算机辅助分析外源DNA环境下(不同孵育时间、DNA长度、DNA浓度)真鲷精子活率和运动速度的变化,进一步采用PCR和荧光探针的方法检测外源DNA与真鲷精子的结合情况,并通过人工授精来评价外源DNA能否转染精子并传递至F0代。结果表明:外源DNA浓度、孵育时间及DNA长度对精子活率无显著影响。在5μg/106个精子的高浓度10 kb DNA下孵育12 h,真鲷精子仍具有(75.89±5.55)%的精子活率,平均直线速度(70.97±6.37)μm/s,与对照组无显著差异,但精子平均曲线速度显著下降,比对照组低21.85μm/s。真鲷精子与带Fluorescein荧光素的DNA片段共孵育后,部分精子在荧光显微镜下观察到绿光。将DNA孵育后精子进行人工授精表明经过1μg DNA/106个精子孵育后的受精率和孵化率无显著下降,通过PCR法并没有在外源DNA处理的精子和F0代中检测到目的基因,表明外源DNA虽然能够吸附在真鲷精子表面,但并不足以携带进入卵子产生转基因后代。

The effect of exogenous DNA （（DNA concentrations, DNA length and incubation time）） on the motility of red seabream （Pagrus major） sperm was studied using computer-assisted sperm analysis （CASA）. Sperm fertility was evaluated by artificial insemination （AI）. The polymerase chain reaction （PCR） and sperm treatment with Fluorescein-DNA were performed to assess the ability of spermatozoa to bind and internalize exogenous DNA. There was no significant difference in the percentage of motile sperm （MOT） between the DNA treated group and control group. The MOT was maintained at （75.89±5.55）% after treatment with high concentrations of DNA （5μg/106 spermatozoa）, whereas the curvilinear velocity and straight line velocity of sperm were decreased by 30.78μm/s and 20.52μm/s, respectively. Green fluorescence was observed in sperm incubated with Fluorescein-DNA, however, no transgene signal was found in DNA treated sperm and embryos after AI through PCR. This result indicates that the exogenous DNA binding to the sperm of red seabream can＇t be carried into the egg and transgenic offspring.