两种提取枝角类休眠卵基因组DNA方法的比较

Comparison of two methods for extracting DNA of resting eggs by Cladocera

休眠卵是枝角类在环境胁迫条件下通过有性生殖形成的,休眠卵的孵化对种群补充与恢复有着重要作用.沉积于底泥中的休眠卵保存了不同阶段的种群遗传信息,有效地提取枝角类休眠卵基因组DNA是进一步研究水体枝角类遗传多样性的关键环节.采用目前国际上使用的氯仿异戊醇提取法和玻璃乳提取法分别对流溪河水库底泥中表层和底层的盔型溞(Daphnia galeata)休眠卵进行基因组DNA提取的比较分析.结果显示,氯仿异戊醇提取法成功率为27.5%,平均浓度为14.25±1.84 ng/μl;玻璃乳提取法成功率为65.0%,平均浓度为28.37±2.56 ng/μl.无论是提取成功率还是提取浓度玻璃乳提取法都显著高于氯仿异戊醇提取法,且玻璃乳提取法所用试剂少,不涉及有毒试剂,操作步骤简单,整个提取过程所用时间短,玻璃乳提取法提取枝角类休眠卵基因组DNA是一种快捷实用的方法.

Most freshwater cladocerans produce resting eggs in their life cycle under harsh conditions. The hatchings from those eggs not only impact cladoceran population and community succession but also link to the longterm shifts in cladoceran genetic structure over time. DNA extraction from resting eggs became a key to enable genetic analyses of past cladoceran populations. In order to find a suitable method for extracting high quality genomic DNA from Cladocera resting eggs, the Uhra-Sep Beads method and chloroform isoamyl alcohol method were used to extract DNA from resting eggs of Daphnia galeata in Liuxihe Reservoir. The success rate and concentration of the DNA extracted by Ultra-Sep Beads method were higher than those from chloroform isoamyl alcohol method. The success rate and concentration of the DNA extracted by Uhra-Sep Beads method reach to 65.0% and 28.37 ± 2.56 ng,/μl, while 27.5% and 14.25± 1.84 ng/μl by chloroform isoamyl alcohol method, respectively. The comparative study exhibited that Uhra-Sep Beads method is an effective method of DNA extraction for resting egg of Cladocera in the sediment, given its simple and fast process and avoiding using chloroform.