【摘要】 为了探究不同提取方法对葱属蔬菜作物叶绿体DNA提取效果的影响,首先以大葱叶片为实验材料,采用高盐低p H值法和蔗糖DNase法,对其进行叶绿体DNA提取测试。结果表明,高盐低pH法优于蔗糖DNase法,其DNA纯度相似,A260/A280值分别1.984和1.977;产率差异大,分别为0.482μg/g FW (fresh weight, FW)和0.059μg/g FW,高盐低pH值法是蔗糖DNase法产率的8.17倍。随后,利用叶绿体MatK、线粒体Atp6和核LFY基因设计引物,根据各组引物自身的扩增效率,以试剂盒提取的总DNA为对照,分别对三种细胞器DNA拷贝数的比值进行评估研究,高盐低pH法提取叶绿体DNA含量是试剂盒法的78.38倍,而蔗糖DNase法为15.51倍,高盐低pH法是蔗糖DNase法的5.05倍。高盐低pH法提取的叶绿体DNA产率和含量均优于蔗糖DNase法,同时在此基础建立了细胞器DNA含量定量分析方法。随后我们用高盐低pH法对葱属蔬菜作物的洋葱、大蒜和韭菜进行了测试,获得了预期的效果。该研究为今后葱属作物高效、高纯度、无污染的叶绿体DNA提取和葱属蔬菜作物细胞器全基因组测序提供依据。更多还原

【Abstract】 In order to investigate the effect of different extraction methods on the extraction of chloroplast DNA(cpDNA) from Allium vegetable crop, the blades of Allium fistulosum were selected as the material. High-salt Low-pH(HSLp) method and Sucrose DNase(SucDNase) method were applied to extract the chloroplast DNA from Allium fistulosum. The results showed that HSLp method was superior to SucDNase method. These two methods acquired similar DNA purity, and the A260/A280 values were 1.984 and 1.977, respectively. However, in DNA yields,HSLp method was 0.482 μg/g FW(fresh weight, FW) and SucDNase method was 0.059 μg/g FW. The yield rate of HSLp method was 8.17 times higher than SucDNase method. Subsequently, primers were designed based on MatK,mitochondrial Atp6 and nuclear LFY gene. According to the amplification efficiency of the primers, the ratio of DNA copy numbers of the three organelles was evaluated by the total DNA extracted from the kit. The extractedDNA content by cpDNA by HSLp method was 78.38 times higher than Kit method, and 15.51 times higher than SucDNase method. HSLp method was 5.05 times of SucDNase method. The chloroplast DNA yield rate and content extracted by HSLp method were both better than SucDNase method. At the same time, the quantitative analysis method of organelle DNA content was established based on this. Then we evaluated Allium cepa, Allium sativum and Allium tuberosum Rottl.ex spr. using the HSLp method, and obtained the expected results. This study could provide a basis for the extraction of cpDNA from Allium crops with high efficiency, high purity and pollution-free, which could offer reference for further whole genome sequencing of organelle in Allium crops.更多还原