摘要
为解决昆虫样本在保存和运输过程中因DNA降解而导致DNA提取效果差的问题。研究采用0.9%NaCl溶液、Tris-EDTA盐水缓冲液(STE)、磷酸缓冲盐溶液(PBS)、无菌水对朽木甲酒精浸制标本进行预处理,提取基因组DNA后,比较分析基因组DNA的质量、浓度及PCR的扩增效果。结果表明:0.9%NaCl溶液、STE缓冲液、PBS缓冲液这3种预处理方式均可在一定程度上提升朽木甲酒精浸制标本DNA的得率和PCR扩增的质量,其中使用PBS缓冲液进行预处理对朽木甲酒精浸制标本DNA提取的综合提升效果最佳;使用无菌水进行预处理对样本DNA提取没有任何提升作用,反而会使PCR扩增的效果更差;相较于线粒体基因片段的PCR扩增,除无菌水外的其他3种预处理方式对核基因的PCR扩增提升效果均较佳;此外,不同预处理均对提取到的DNA的OD_(260/280)值没有显著的影响。本研究结果为朽木甲酒精浸制标本的基因组DNA提取提供了更好的预处理方式,大大提高了其DNA提取的得率和扩增质量,减少了在DNA提取过程中标本数量的消耗和浪费。为后续的分子研究工作奠定了基础,同时也为其他昆虫酒精浸制标本的DNA提取方案提供了优化思路。
In order to solve the problem of poor DNA extraction effect due to DNA degradation during storage and transportation of insect samples,this study adopted 0.9% NaCl buffer,Saline Tris-EDTA(STE),phosphatebuffered saline(PBS),and sterile water to pretreat the specimens of comb-clawed beetle,and then extracted the insect genomic DNA.By comparing and analyzing the quality and concentration of insect genomic DNA and the amplification effect of PCR,the results show that the three pretreatment methods of 0.9%NaCl buffer,STE buffer and PBS buffer can improve the DNA yield and PCR extraction of DNA from alcohol-impregnated specimens of Comb-clawed beetle;Pretreatment amplification with PBS buffer has the best comprehensive improvement effect while sterile water does not improve the extraction of sample DNA,and the PCR amplification effect is worse;Compared with the PCR amplification of mitochondrial gene fragments,the three pretreatment methods except sterile water have a better effect on the PCR amplification of nuclear genes;In addition,different pretreatments have no significant effect on the DNA OD_(260/280)value of the extracted DNA.The results provide a better pretreatment method for the extraction of genomic DNA from alcohol-impregnated specimens of comb-clawed beetle,greatly improving the yield and quality of DNA extraction and reducing the consumption and waste of the number of specimens in the DNA extraction process.This study lays a foundation for the molecular research in the future,and also provides an optimization idea for other DNA extraction schemes of alcohol-impregnated insect specimens.
作者
刘鹏冀
王文强
苑彩霞
LIU Pengji;WANG Wenqiang;YUAN Caixia(School of Life Sciences,Yan’an University;Engineering and Technological Research Center for Conservation and Utilization of Regional Biological Resources,Yan’an 716000,China)
出处
《延安大学学报（自然科学版）》
2022年第2期83-88,共6页
Journal of Yan'an University：Natural Science Edition
基金
国家自然科学基金项目(31960113)
陕西省林业局项目(SXRCZB2019-ZC-DY1002)
延安大学研究生教育创新计划项目(YCX2020076)。
