一种适用于PCR扩增的甜菜干种子DNA快速提取方法的研究

The Study on Rapid Extraction Method of DNA in Dry Beet Seeds for PCR

为了寻求一种快速提取甜菜DNA的方法,不经液氮研磨,利用改良CTAB法对甜菜干种子进行了DNA的提取,通过降低水浴时间及利用冰浴快速析出DNA,减少了提取的时间。提取的DNA经0.8%的琼脂糖检测,DNA条带完整,没有降解,经与λDNA进行比对,每毫克干种子可提取DNA大约50 ng,利用SSR引物BvATT1及SRAP引物组合(me1,em2)对提取的DNA进行扩增,用6%的非变性聚丙烯酰胺进行检测,结果条带清晰,特异性强。试验结果表明,利用改良CTAB法完全可以从甜菜干种子中提取到符合甜菜分子试验要求的DNA。本研究首次利用改良CTAB法从甜菜干种子中提取到了高质量的DNA,建立了快速提取甜菜DNA的体系,为分子标记将来在甜菜品种指纹图谱构建及辅助育种等方面的应用奠定了坚实的基础。

To find a way to extract DNA of beet rapidly, modified CTAB method was used for DNA extraction from beet＇ s dried seeds without liquid nitrogen grinding. It reduced time of water bath and had DNA separated out rapidly by ice bath, which reduced time of extraction. With 1% agarose testing for extracted DNA, the bands were intact without degradation. About 50 ng DNA could be extracted from 1 mg dried seeds by contrasting with gDNA. The extracted DNA was amplified using combination of SSR primer BvATT1 and SRAP primer （me1, em2） and the amplified product was detected with 6% non-denaturing polyacrylamide. The result showed that the bands were clear and specific. The result suggested that the DNA which met the requirements of beet molecular experiments could be definitely extracted from dried beet seeds with CTAB method. This research firstly utilized modified CTAB method to extract high-quality DNA from dried beet seeds and establishes the system of extracting beet DNA rapidly, which laid the solid foundation for future application of molecular marker in DNA fingerprint establishment and assistant breeding of beet species.