菊芋叶绿体分离方法比较研究  

Comparison of Chloroplast DNA Extraction Methods from Jerusalem artichoke

作　　者：许盼盼 杨世鹏[1] 王丽慧[1] 孙雪梅[1,3] 高洁铭 李莉[1,3] 钟启文[1,3] XU Panpan;YANG Shipeng;WANG Lihui;SUN Xuemei;GAO Jieming;LI Li;ZHONG Qiwen(Qinghai Vegetable Genetics and Physiology Laboratory,Qinhai University,Xining 810016,China;College of Agriculture and Animal Husbandry,Qinghai University,Xining 810016,China;State Key Laboratory of Plateau Ecology and Agriculture,Qinghai University,Xining 810016,China)

机构地区：[1]青海大学农林科学院青海省蔬菜遗传与生理重点实验室,西宁810016 [2]青海大学农牧学院,西宁810016 [3]青海大学三江源生态和高原农牧业国家重点实验室,西宁810016

出　　处：《西北农业学报》2019年第4期657-663,共7页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31460523;31660569;31660588);青海省科技厅项目(2016-ZJ-751)~~

摘　　要：以菊芋叶片为材料,通过比较提取其叶绿体DNA的5种方法(改良高盐-低pH法、Percoll密度梯度离心法、GENMED试剂盒物理法、GENMED试剂盒化学法及DNaseⅠ差速离心分离法),筛选出适合菊芋叶绿体NDA的提取方法。利用显微镜、核酸蛋白测定仪、琼脂糖凝胶电泳及菊芋基因特异引物RAPD检测。结果显示:改良高盐-低pH法分离得到的叶绿体在40×物镜下其数目多,浓度高,背景清晰,提取菊芋叶片cpDNA的OD_(260)/OD_(280)为1.926,质量浓度为295.63μg/μL。经10 g/L琼脂糖凝胶电泳检测发现,cpDNA条带清晰整齐,无核DNA污染;且提取的cpDNA通过4对引物均能扩增到片段。结果表明,改良高盐-低pH法能够快速获得菊芋高质量的叶绿体DNA。In order to screen out the chloroplast DNA(cpDNA) extraction method suitable for Jerusalem artichoke chloroplast DNA.In this experiment,Jerusalem artichoke leaves were used as materials to compare the modified high salt-low pH method,Percoll density gradient centrifugation method,GENMED kit physics method,GENMED kit chemical method and DNaseⅠ differential centrifugation method. The DNA quality of Jerusalem artichoke chloroplast was determined by microscope,nucleic acid protein analyzer,agarose gel electrophoresis and Jerusalem artichoke gene-specific primer RAPD. The results showed that the chloroplasts isolated by the modified high-salt-low pH method had a large number,high concentration and clear background under the 40× objective lens. The OD260/OD280 of cpDNA extracted from Jerusalem artichoke leaves was 1.926,and the mass concentration was 295.63 μg/μL. After 10 g/L agarose gel electrophoresis,the cpDNA band was clear and neat,and there was non udear DNA contamination.The extracted cpDNA could amplify the fragment by 4 pairs of primers.The results showed that the improved high-salt-low pH method could quickly obtain the high quality chloroplast DNA of Jerusalem artichoke,which laid a foundation for the study of the chloroplast genome of Jerusalem artichoke and even the compositae.

关 键 词：改良高盐-低pH法 叶绿体基因组提取 叶绿体分离 菊芋 

分 类 号：S649[农业科学—蔬菜学]