葱属蔬菜作物叶绿体DNA提取及其质量评价体系的建立  

Chloroplast DNA Extraction of Allium Vegetable and Establishment of Quality Evaluation System

作　　者：孙雨晴[1,2] 陈立 杨亚会 杨妍妍[2] 吴雄 于占东 霍雨猛 Sun Yuqing;Chen Li;Yang Yahui;Yang Yanyan;Wu Xiong;Yu Zhandong;Huo Yumeng(College of Horticulture,Jilin Agricultural University,Changchun,130118;Vegetables and Flowers Research Institute,Shandong Academy of Agri-cultural Sciences,Shandong Provincial Key Laboratory for Biology of Greenhouse Vegetable,Shandong Branch of National Improvement Center for Vegetable,Ji＇nan,250100;College of Horticulture Science and Engineering,Shandong Agricultural University,Taian,271018)

机构地区：[1]吉林农业大学园艺学院,长春130118 [2]山东省农业科学院蔬菜花卉研究所山东省设施蔬菜生物学重点实验室国家蔬菜改良中心山东分中心,济南250100 [3]山东农业大学园艺科学与工程学院,泰安271018

出　　处：《分子植物育种》2018年第20期6802-6807,共6页Molecular Plant Breeding

基　　金：山东省农业科学院青年英才培养计划(IVFSAAS2016-2018-01);国家自然科学基金(31672165; 31201635;31301786)共同资助

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。In order to investigate the effect of different extraction methods on the extraction of chloroplast DNA （cpDNA） from A llium vegetable crop, the blades of A llium 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 A llium fistulosum. The results showed that HSLp method was superior to SucDNase method. These two methods acquired similar DNA purity, and the A 260/A 280 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 MotK, mitochondrial A tp6 and nuclear LFF gene. According to the amplification efficiency of the primers, the ratio of DNA copy numbers of the three organeUes was evaluated by the total DNA extracted from the kit. The extracted DNA 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 A llium cepa, A llium sativum and A llium 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 Alliurn crops with high efficiency, high purity and pollution-free, which could offer reference for further whole genome sequencing of organelle in A llium crops.

关 键 词：葱属蔬菜作物 叶绿体DNA 高盐低pH值法 蔗糖DNase法 

分 类 号：S633[农业科学—蔬菜学]