| 菊芋叶绿体分离方法比较研究 |
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| 引用本文：许盼盼,杨世鹏,王丽慧,孙雪梅,高洁铭,李 莉,钟启文.菊芋叶绿体分离方法比较研究[J].西北农业学报,2019,28(4):657~663 |
| DOI:10.7606/j.issn.1004-1389.2019.04.019 |
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| 基金项目:国家自然科学基金（31460523，31660569，31660588）；青海省科技厅项目（2016-ZJ-751）。 |
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| 中文摘要: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OD260/OD280为1.926，质量浓度为295.63 μg/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。 |
| 中文关键词:改良高盐-低pH法 叶绿体基因组提取；叶绿体分离；菊芋 |
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| Comparison of Chloroplast DNA Extraction Methods from Jerusalem artichoke |
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| Abstract: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. |
| keywords:Modified high salt-low pH method Extraction chloroplast genome Chloroplast separation Helianthus tuberosus L.〖HT6〗 |
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