梅叶绿体分离与测序方法优化  

The Optimization of Separation and Sequencing Methods of Chloroplast in Mei Flower

作　　者：薛松 倪照君[2] 乔玉山[2] 侍婷[2] 高志红[2] XUE Song;NI Zhaojun;QIAO Yushan;SHI Ting;GAO Zhihong

机构地区：[1]南京农业大学,南京210095 [2]南京农业大学园艺学院,南京210095

出　　处：《中国园林》2020年第S01期60-63,共4页Chinese Landscape Architecture

基　　金：农业部种质资源保护项目;科技部农作物种质资源共享平台(编号NICGR2019-98)共同资助.

摘　　要：叶绿体基因组是研究进化关系的理想基因组。为了获得最适合梅(Prunus mume)叶绿体提取及测序方法的方案,以梅品种‘龙眼’新鲜幼叶为试验材料,比较叶绿体试剂盒,高盐-低pH配合Percoll密度梯度以及改良高盐-低pH3种方法分离叶绿体的效果。结果表明改良高盐-低pH法提取梅cpDNA的OD260/OD280值为2.043,质量浓度为5 286.4ng/ul,是3种方法中最适合梅叶绿体提取的方法。此外,通过比较直接与拼接2种测序方法得到的‘龙眼’梅叶绿体基因组差异,发现两者的显著差异为直接测序法测得基因组在IR区112~118kb区间有一段冗余序列,基因组重排相似度高于99%。综合考虑,梅叶绿体最佳提取方法为改良高盐-低pH法,直接测序法获得的信息量较大但增加了提取叶绿体DNA的成本。The chloroplast genome is a perfect genome for studying evolutionary relationships. To obtain the most suitable method for Mei flower chloroplast separation and sequencing, the chloroplast DNA(cpDNA) of Mei flower was extracted by SDS-proteinase K from fresh and tender leaves of Prunus mume ‘Longyan’ by three developed techniques:improved high salt-low pH, high salt-low pH combined with Percoll density gradient and plant chloroplast DNA kit). The results showed that the improved high-salt-low-pH method was the best method for isolation of chloroplasts. OD260/OD280 was 2.043, and the mass concentration was 5 286.4 ng/ul. It is the most suitable method for isolation of chloroplast and extraction of cpDNA from leaves. With the comparison of the two sequencing methods, it was found that direct sequencing had more redundant sequences than reference splicing sequencing in the 112-118 kb of IR region and the similarity of rearrangement detection of two chloroplast genome is more than 99%. In conclusion, the best extraction method of chloroplast in Mei flower is the improved high salt-low-pH method, and the direct sequencing method is more accurate but increases the cost of separating chloroplast.

关 键 词：园林植物 梅 叶绿体DNA提取 改良高盐-低pH法 叶绿体测序 

分 类 号：S688[农业科学—观赏园艺]