一种从雉类孵化卵壳中提取DNA的方法

A Method for DNA Extraction from Incubated Eggshells of Pheasants

从鸟类已孵化的卵壳中提取DNA属于一种非损伤性取样技术,在鸟类分子生态学研究中具有广阔的应用前景。本实验以河南董寨自然保护区白冠长尾雉(Syrmaticus reevesii)和环颈雉(Phasianuscolchicus)已孵化的卵壳为材料,利用红细胞破碎液、蛋白酶K及RNA酶等试剂,对卵壳膜内的总DNA进行了提取,建立了一种提取高质量DNA的新方法。琼脂糖凝胶电泳检测显示,采用新改进的方法提取出的DNA条带清晰。同时,利用聚合酶链式反应(PCR)技术成功地从总DNA中扩增出两种雉类的线粒体DNA控制区(CR)片段,测序后与GenBank中同一物种的CR序列进行比对,结果证实了PCR产物的真实性。文中利用卵壳膜提取出的DNA,对一窝环颈雉的雏鸟进行了性别鉴定,其结果与根据形态特征进行鉴定的结果完全一致,均为3雄4雌,从而证实了从卵壳膜中提取DNA的真实性。该种DNA提取方法在雉类研究中将具有广泛的应用。

Extracting DNA from eggshells is a non-invasive sampling method which has a wide application in the studies of avian molecular ecology. By using red blood cell broken-up solution, proteinase K and RNase, total DNA with high quality was extracted from the egg membranes in the incubated eggshells of two pheasant species, Reeves＇ s Pheasant Syrmaticus reevesii and Ring-necked Pheasant Phasianus colchicus from Dongzhai Nature Reserve in Henan Province. An improved method for extracting DNA from eggshells has been established. The mitochondrial control region （CR） of the two species was successfully amplified and sequenced. The DNA sequences were the same as those published in the Genbank for the same two species, which showed the reliability of the PCR products. At the same time, the sexes of one brood of Ring-necked Pheasant were identified by detetion of the Chromo-helicase- DNA- binding genes. The molecular sexing results of 3 males and 4 females were consistent with those identified by their morphological characteristics. Eggshell has been proved to be an ideal source of DNA extraction and can be widely used in the pheasant studies.