引用本文:	郑君飘,刘清华,徐世宏,马道远,肖志忠,林帆,肖永双,李军.真鲷精子在外源DNA 下的生理特性和转染效果[J].海洋科学,2014,38(7):1-6.

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真鲷精子在外源DNA 下的生理特性和转染效果
郑君飘^1,2, 刘清华¹, 徐世宏¹, 马道远¹, 肖志忠¹, 林帆¹, 肖永双¹, 李军¹
1.中国科学院海洋研究所;2.中国科学院研究生院

摘要:

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5 μg/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1 μg 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。

关键词: 外源DNA 真鲷(Pagrus major) 精子精子介导转基因

DOI：10.11759/hykx20131223002

分类号:

基金项目:国家自然科学基金资助项目(31072212, 41076100); 海洋经济创新发展区域示范项目(12PYY001SF08); 国家科技基础条件平台建设运行项目–水产资源平台运行服务

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

Abstract:

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 (5 μg/10⁶ spermatozoa), whereas the curvilinear velocity and straight line velocity of sperm were decreased by 30.78 μm/s and 20.52 μm/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.

Key words: Exogenous DNA pagrus major sperm sperm-mediated gene transfer