水霉菌总DNA提取方法研究
STUDY ON TOTAL DNA EXTRACTION METHODS OF SAPROLEGNIA
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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。
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关键词:
- 水霉菌 /
- DNA提取 /
- 溶菌酶 /
- 十六烷基三甲基溴化铵(CTAB) /
- 尿素 /
- 十二烷基硫酸钠(SDS)
Abstract: Saprolegnia are parasitic on fish and their eggs and known as the most important pathogens in freshwater fish. Infec2tious diseases caused by water molds can cause losses of freshwater fish in both nature and commercial fish farms. It is very im2portant to obtain the high quality and effective total DNA to study the mechanism of molecular nosogenesis, identify the speciesusing molecular marker and gene diagnoses.In this study, Saprolegnia parasitica, S. ferax, S. dolica, S. sp1and S. spwere used as test materials. The DNA extrac2tion methods of Lysozyme, CTAB, improved CTAB, CH4N2O and SDSwere compared. Firstly, the 30 mg (net weight) of freshlysubcultured mycelium of five different pathogenetic fish fungi were taken to freeze at - 70 e for 30min in 1. 5mL Eppendorftubes, respectively. Then they were transferred to room temperature and grinded using appropriate abrasive tool. This processwas repeated once. Secondly, the trituration of five mycelium were digested by lysozyme digestion, CTAB digestion, improvedCTAB digestion, CH4N2O digestion and SDS digestion, respectively, then extracted DNA using relevant processes. The yield andquality of DNA were evaluated with ultraviolet radiation spectrophotometer and PCR of the ITS rDNA. The result of ultraviolet ra2diation spectrophotometer showed that DNA can be obtained using five different methods and the genomic DNA extracted with im2proved CTABmethod had the best quality and yield, the A260/ A280 ratio was between 1. 79) 1. 82 and the concentrat ion ofDNAwas about 45Lg/mL. The result of PCR showed that fiveDNA samples extracted with improved CTABmethod can bewell ampli2fied and obtained the bright, no tail and trim electrophoresis strip. However, there were unclear or vacant electrophoresis stripsin the DNA samples extracted with other methods. Therefore, the DNA extraction method of improved CTABwas the best methodto gain the DNA of Saprolegnia for the study of molecular level.-
Keywords:
- Saprolegnia /
- DNA extraction /
- CTAB /
- Lysozyme /
- CH4N2O /
- SDS
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