富含多糖的转基因石斛基因组DNA提取方法(英文)

Study on DNA Isolation from Polysaccharides-rich Transgenic Dendrobium

在石斛兰转基因研究中,需通过PCR和Southern杂交等手段检测外源基因是否转入并整合到转化植株基因组。由于转化石斛植株生长缓慢,且含有多糖,因此从转化植株中提取高质量的基因组DNA以尽快对转基因苗进行分子检测存在较大困难。本研究旨在通过改良现有的DNA提取法(CTAB法或SDS法),克服转化石斛植株基因组DNA提取中碰到的产量低,纯度不高而导致难以进行PCR或酶切等问题。在本研究所用的3种改良法中,方法Ⅱ能从少量的转化石斛苗中提取出高产量和高纯度的基因组DNA。研究结果表明方法Ⅱ提取的基因组DNA完全适用于转基因石斛的外源基因PCR扩增,限制性酶切和Southern杂交分析。

Isolation of high-quality genomic DNA of transgenic Dendrobium plantlets for rapid screen or genetic analysis by PCR amplification and Southern blot is difficult, for the transgenic plantlets grow very slowly and contain high-level polysaccharides. In order to overcome such problems as low yield, degradation, poor PCR amplification and poor restriction digestion, three methods developed from hexadecyltrimethylammonium bromide （CTAB） or sodium dodecyl sulfate （SDS） method respectively were optimized and compared in this paper. Of the three modified methods used, method Ⅱ produced pure and high-quantity genomic DNA from small amount samples oftransgenic Dendrobium or other plantlets in vitro tissue culture. DNA isolated by method Ⅱ was proved amenable to PCR amplification, restriction digestion and Southern blot analysis of transgenic De ndrobium plantlets.