摘要
本实验采用EP管反复冻融和研磨的破壁方式,利用溶菌酶法、CTAB法、改良CTAB法、尿素法、十二烷基硫酸钠(SDS)法等5种方法,分别对5种鱼类致病性水霉菌(寄生水霉、多子水霉、异株水霉及两未定种)的基因组DNA进行提取,并采用紫外分光光度计和ITS区基因(包括5.8SrDNA)PCR扩增对DNA进行了评价。紫外分光光度计检测结果表明,5种方法均可提取到水霉菌DNA,其中改良CTAB法提取的5种水霉菌的DNA产量和质量最高,A260/A280在1.79—1.82之间,浓度为45μg/mL;PCR检测结果表明,只有改良CTAB法提取的DNA全部扩增到明亮、整齐、无拖尾的特异性条带,其他几种方法均存在暗带或无带现象。因此,改良CTAB法可以作为水霉菌DNA提取以开展分子生物学研究的首选方法。
Saprolegnia are parasitic on fish and their eggs and known as the most important pathogens in freshwater fish. Infectious diseases caused by water molds can cause losses of freshwater fish in both nature and commercial fish farms. It is very important to obtain the high quality and effective total DNA to study the mechanism of molecular nosogenesis, identify the species using molecular marker and gene diagnoses.
In this study, Saprolegnia parasitica, S.ferax, S.dolica, S.sp1 and S.sp2 were used as test materials. The DNA extraction methods of Lysozyme, CTAB, improved CTAB, CH4N2O and SDS were compared. Firstly, the 30 mg （net weight） of freshly subcultured mycelium of five different pathogenetic fish fungi were taken to freeze at -70℃ for 30min in 1.5mL Eppendorf tubes, respectively. Then they were transferred to room temperature and grinded using appropriate abrasive tool. This process was repeated once. Secondly, the trituration of five mycelium were digested by lysozyme digestion, CTAB digestion, improved CTAB digestion, CH4N2O digestion and SDS digestion, respectively, then extracted DNA using relevant processes. The yield and quality of DNA were evaluated with ultraviolet radiation spectrophotometer and PCR of the ITS rDNA. The result of ultraviolet radiation spectrophotometer showed that DNA can be obtained using five different methods and the genomic DNA extracted with improved CTAB method had the best quality and yield, the A260/A280 ratio was between 1.79—1.82 and the concentration of DNA was about 45μg/mL. The result of PCR showed that five DNA samples extracted with improved CTAB method can be well amplified and obtained the bright, no tail and trim electrophoresis strip. However, there were unclear or vacant electrophoresis strips in the DNA samples extracted with other methods. Therefore, the DNA extraction method of improved CTAB was the best method to gain the DNA of Saprolegnia for the study of molecular level.
出处
《水生生物学报》
CAS
CSCD
北大核心
2008年第1期68-73,共6页
Acta Hydrobiologica Sinica
基金
湖北省科技攻关计划项目(2007AA203A01)
中国科学院领域前沿项目(053903)
