不同提取方法和不同组织对中华绒螯蟹 DNA提取效果的比较研究

为了优化中华绒螯蟹基因组DNA提取方法,采用酚-氯仿法和试剂盒法分别对中华绒螯蟹的鳃、肌肉、血淋巴液和刚毛4种组织进行DNA提取,并从纯度、浓度、片段完整性及微卫星标记PCR扩增效果4个方面评价提取DNA的质量.结果显示:酚-氯仿法提取的DNA片段比试剂盒法提取的更完整,且DNA浓度较高,但纯度稍低;无论何种提取方法,刚毛和血淋巴液中的DNA纯度都高于鳃和肌肉,4种组织中的DNA含量依次为鳃＞肌肉＞刚毛＞血淋巴液,未剪碎刚毛中未能提取到基因组DNA;微卫星标记的电泳结果表明,两种提取方法下4种组织中提取的DNA均可满足微卫星标记的应用要求.综上,本研究建立了一种简便的非损伤性中华绒螯蟹的采样及其DNA提取方法,这对于进行中华绒螯蟹遗传育种和分子标记研究等方面具有一定的积极作用.

Comparative study on extraction of genome DNA from different tissues of Chinese mitten crab (Eriocheir sinensis) by dissimilar methods

To optimize genome DNA extraction method of Chinese mitten crabs, genome DNA was extracted from gills, muscle, blood and bristles of Chinese mitten crabs using phenol -chloroform method and DNA extraction kit, and the quality of these DNA samples was evaluated in four aspects: the purity, concentration, fragment completeness and microsatellite marker PCR amplification effect. The results showed that, compared with the method of DNA extraction kit, the DNA extracted by phenol-chloroform method was much more complete, had higher concentration, but lower purity. Both the extraction methods showed that the purity of DNA in bristles and blood were higher than that in the gills and muscle, and the descending order of DNA content was gill, muscle, bristles, blood, while the uncutting bristles were failed to extract genomic DNA. Microsatellite PCR electrophoresis results showed that the DNA extracted from four different tissues by two dissimilar DNA extraction methods could meet the requirements of the application of microsatellite markers. In conclusion, the present study established a convenient noninvasive sampling method and a procedure of DNA extraction that could promote genetic breeding and molecular marker research of E. sinensis.