电洗脱法和透析在紫菜腐霉PacBio全基因组测序DNA样品纯化中的应用  

The Applications of Electroelution and Dialysis to Purify Genome DNA of Pythiumporphyrae

作　　者：邹丹丹[1,2] 唐祥海[1,2] 莫照兰[3] 茅云翔[1,2] ZOU Dan-Dan TANG Xiang-hai MO Zhao-Lan MAO Yun-Xiang(The Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China College of Marine Life sciences Ocean University of China, Qingdao 266003, China Yellow Sea Fisheries Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China)

机构地区：[1]中国海洋大学海洋生物遗传学与育种教育部重点实验室,山东青岛266003 [2]中国海洋大学海洋生命学院,山东青岛266003 [3]农业部渔业资源可持续发展重点实验室,中国水产科学研究院黄海水产研究所,山东青岛266071

出　　处：《中国海洋大学学报（自然科学版）》2017年第11期47-52,共6页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(31372517);国家高技术研究计划项目(2012AA10A406);山东省自主创新专项(2013CXC80202)资助~~

摘　　要：紫菜腐霉(Pythiumporphyrae)是一种专性侵染紫菜并引起紫菜赤腐病的病原菌。为了深入了解紫菜腐霉的遗传信息,本文计划对其开展全基因组测序,而制备高质量的DNA样品是关键性工作。海洋生物生境和代谢的特殊性,细胞中富含的一些次级代谢物质往往影响着DNA样品的质量。紫菜腐霉的基因组DNA样品中具有严重的RNA污染,即使通过延长RNase A降解时间和增加降解次数也不能完全消除其中的RNA,无法获得满足于PacBio文库构建和测序的DNA样品。本研究首先通过琼脂糖凝胶电泳(Agarosegel electrophoresis,AGE)来区分DNA和RNA,然后将DNA条带所在的胶块切下并放入透析袋中继续电泳,使DNA从胶条中迁移到透析袋内的缓冲液中,从而获得DNA的TAE溶液。然后通过透析的方法去除DNA的TAE溶液中的乙酸钠,最终获得DNA的TE溶液。经AGE检测发现DNA条带清晰、无RNA污染;经脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)检测发现DNA完整性较好,可用于PacBio文库的构建和测序。PythiumPorphyraeis a special pathogen to infect Nori,which caused red rot disease in Pyropiayezoensisfrequently.It brings seriously reduce in Nori production and quality every year in China,Korea and Japan.So,more genetic information about the pathogen could help us exploit disease prevention and control measures.The most useful and quickly method to get the genetic information is carrying on its whole genome analysis.High quality DNA sample is the key work for carrying on the project.Particular habitation and metabolism and abundant secondary metabolites in cells of marine organism often affects the quality of the genome DNA.The P.porphyraegenome DNAwas polluted by RNA seriously in our preliminary experiment.Usually,RNA was easily degradation by RNase A,but the RNA which residual in P.porphyrae genome DNA was not removed absolutely,even by extending RNase A degradation time and increasingRNase A degradation times,RNA pollution in the DNA simple cannot be completely degradation,the DNAlost and degradation seriously,butthe DNA simple qualitystill cannot reach up to the standard for constructing PacBio libraries.The librarywhich was constructed mandatory cannot sequence normally.In this study,DNA was obtained by EDTA methods,then the agarosegel electrophoresis（AGE）was used to separate genome DNA and RNA.The DNA bandswere cut under UV light quickly andwere transferredinto dialysis bag,further electrophoresisto insure the direction of the DNA band migrating as before.Stopping electrophoresis after the DNA band disappeared from the agarose gelby checking under UV light.This result showed that the DNA in agarose gel had enteringto the TAE solution of the dialysis bag absolutely.The dialysis which contained DNA and agarose gel,was put into 1liter of TE buffer,the sodium acetate（CH3COONa）in the dialysis would moved from the dialysis bag to the TE buffer.At last,the pure TE solution of DNAwas got eventually.Enriching DNA by anhydrous ethanol and then detecting the DNA by AGE and pulsed field gel electrophoresis（

关 键 词：紫菜腐霉 基因组DNA RNA污染 透析 

分 类 号：Q556.2[生物学—生物化学]