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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。
In the dendrobium transgene research, we need to detect whether the foreign gene is transferred into the genome of the transformed plant by means of PCR and Southern hybridization. Due to the slow growth of Dendrobium plants and the presence of polysaccharides, it is very difficult to extract high quality genomic DNA from transformed plants for molecular detection of transgenic plants as soon as possible. The aim of this study is to overcome the problems of low yield, low purity and difficulty of PCR or digestion in the extraction of genomic DNA from the transformed Dendrobium by improving the existing DNA extraction method (CTAB method or SDS method). Among the three improved methods used in this study, Method II extracted genomic DNA of high yield and purity from a small amount of transformed Dendrobium seedlings. The results showed that the genomic DNA extracted by the method Ⅱ was completely suitable for the PCR amplification of exogenous genes, restriction analysis and Southern hybridization analysis.