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• 技术与方法 • 上一篇    

福尔马林固定铜鱼基因组DNA的提取与扩增

王丹1,2;谭德清1;童金苟1   

  1. 1中国科学院水生生物研究所淡水生态与生物技术国家重点实验室, 武汉 430072;2中国科学院研究生院, 北京 100039
  • 收稿日期:2008-08-05 修回日期:1900-01-01 出版日期:2009-03-10 发布日期:2009-03-10

Extraction and amplification of genomic DNA from formalin-fixed Coreius heterodon.

WANG Dan1,2;TAN De-qing1;TONG Jin-gou1   

  1. 1State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;2Graduate University of Chinese Academy of Sciences, Beijing 100039, China
  • Received:2008-08-05 Revised:1900-01-01 Online:2009-03-10 Published:2009-03-10

摘要: 铜鱼(Coreius heterodon)作为长江中、上游的重要经济鱼类是研究三峡大坝阻隔效应的重要材料之一。然而,过去的铜鱼标本都保存在福尔马林溶液中,有必要探讨从福尔马林固定的铜鱼标本中有效提取基因组DNA的方法以及这些DNA用于微卫星和线粒体分析的可行性。本实验通过改进的酒精梯度浸泡法去除标本中的甲醛,然后用酚-氯仿抽提法成功地提取到了铜鱼标本的基因组DNA;设计引物后进行了线粒体和微卫星的PCR扩增,扩增产物经银染检测。微卫星扩增结果显示只有部分个体可以扩出目的带,而线粒体控制区部分区段在所有个体中均能稳定重复的扩出;mtDNA SSCP分析显示带型一致。结果表明,福尔马林固定的铜鱼标本可以被用来开展短片段的扩增和遗传变异分析等方面的相关研究。

关键词: 亚高山针叶林, 云杉, 红桦, 萌发, 光照, 温度

Abstract: Coreius heterodon is an important commercial fish species in the upper and middle reaches of Yangtze River, and one of the appropriate fishes for the study of possible ecological effects of the Three Gorges Dam. However, the museum C. heterodon specimens were preserved in formalin. It is necessary to approach the methods of effectively extracting the genomic DNA from formalin-fixed C. heterodon, and the feasibility of using the extracted DNA in microsatellite- and mitochondrial analyses. In the present paper, the genomic DNA of ten formalin-fixed C. heterodon specimens was successfully extracted by the steps of removing the formaldehyde through improved gradual dehydration first and extracting the genomic DNA by the traditional phenol-chloroform method then. After primers designed, the PCR amplification was carried out, and a 205 bp of the control region of mtDNA from all of the ten specimens was successfully amplified. SSCP analysis indicated that no polymorphism was detected in this short fragment, and SSR also showed monomorphic. It was suggested that by using alcohol treatment, the formalin-fixed specimens of C. heterodon could be used as the potentially suitable biological materials in the studies of the possible changes of genetic diversity after large hydropower projects were constructed.

Key words: Subalpine coniferous forest, Picea asperata, Betula albo-sinensis, Germination, Light, Temperature