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以尾叶桉叶片为材料,用高盐低pH法纯化叶绿体,再用SDS法提取叶绿体DNA(cpDNA)。经琼脂糖电泳检测,纯化回收离心力为200 g、300 g、400 g、500 g得到的cpDNA条带较亮、无降解拖尾。以400 g提取的cpDNA为模板,用核基因组及叶绿体基因组特异引物做PCR,能扩增到叶绿体基因片段且没有扩增到核基因片段。以来自尾叶桉白色愈伤组织的核DNA作对照,能扩增到核基因片段且没有扩增到叶绿体基因片段。结果表明:高盐低pH法提取的尾叶桉cpDNA纯度高,没有核基因污染,适用于基因扩增。本研究操作简便,成本低,能为木本植物cpDNA的提取提供参考。
The leaves of Eucalyptus urophylla were used as material to purify chloroplast by high salt and low pH method, then chloroplast DNA (cpDNA) was extracted by SDS method. After agarose gel electrophoresis, the cpDNA bands with the centrifugal force of 200 g, 300 g, 400 g, 500 g were recovered and brightened without degradation. The cpDNA extracted from 400 g was used as a template to amplify the chloroplast gene fragment and did not amplify the nuclear gene fragment by PCR with nuclear genome and chloroplast genome-specific primers. Using nuclear DNA from white callus of Eucalyptus urophylla as a control, it was able to amplify to nuclear gene fragments and not to chloroplast gene fragments. The results showed that the cpDNA of Eucalyptus urophylla, extracted by high salt and low pH, had high purity and no nuclear gene contamination, which was suitable for gene amplification. This study is simple, low cost and can provide reference for the extraction of cpDNA from woody plants.