鼠曲草简单重复序列间扩增反应体系的建立及优化
投稿时间:2019-09-12  修订日期:2019-10-21  点此下载全文
引用本文:江圣圭,董志颖,李卿,赵英魁,黄豆豆,孙连娜.鼠曲草简单重复序列间扩增反应体系的建立及优化[J].药学实践杂志,2020,38(1):42~46,62
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作者单位E-mail
江圣圭 福建中医药大学药学院, 福建 福州 350122  
董志颖 上海中医药大学中药学院, 上海 201203  
李卿 海军军医大学附属长征医院药学部, 上海 200003  
赵英魁 海军军医大学药学院, 上海 200433  
黄豆豆 海军军医大学药学院, 上海 200433  
孙连娜 福建中医药大学药学院, 福建 福州 350122
上海中医药大学中药学院, 上海 201203 
sssnmr@163.com 
基金项目:国家自然科学基金项目(81373954),上海市科委资助课题项目(13ZR1448800)
中文摘要:目的 通过建立并优化鼠曲草简单重复序列间扩增(ISSR-PCR)反应体系,为鼠曲草后续遗传多样性研究提供实验依据。方法 本研究首先采用单因素实验法和全面实验法优化鼠曲草ISSR-PCR反应体系,然后在最适体系下,分步进行引物及其退火温度的筛选,最后验证该体系的可行性。结果 ISSR-PCR最适反应体系包括10 μl Premix Taq DNA聚合酶、0.3 μmol/L引物、10 ng DNA模板、灭菌水加至20 μl;从100条通用引物中最终筛选出10条引物;验证结果表明该体系稳定性高、重复性好,且选定引物多态性好。结论 本研究首次建立了鼠曲草ISSR-PCR扩增体系,并筛选出合适的引物,为鼠曲草后续遗传多样性研究夯实了基础。
中文关键词:鼠曲草  简单重复序列间扩增  体系优化  引物筛选
 
Establish and optimization of inter-simple sequence repeat PCR reaction system of Gnaphalium affine
Abstract:Objective To provide the experimental basis for the subsequent genetic diversity research through establishing and optimizing the inter-simple sequence repeat PCR (ISSR-PCR) reaction system of Gnaphalium affine.Methods The single-factor experimental method and full experimental method were used to optimize the ISSR-PCR reaction system of Gnaphalium affine. Under the optimal system, after screening primers and corresponding annealing temperatures, the systematic feasibility was verified.Results The optimal ISSR-PCR reaction system was consisted of 10 μl Premix Taq DNA polymerase, 0.3 μmol/L primer, 10 ng DNA template, and sterilized water added to 20 μl. Finally, 10 primers were screened from 100 universal primers, and verification results indicated the system had high stability, good reproducibility, and the selected primers had good polymorphism.Conclusion The ISSR-PCR amplification system of Gnaphalium affine was established for the first time and the primers with appropriate annealing temperatures were filtered out, which provided a reference for the subsequent genetic diversity research of Gnaphalium affine.
keywords:Gnaphalium affine  inter-simple sequence repeat PCR  system optimization  primer screening
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