宽叶缬草SRAP-PCR反应体系的优化及引物筛选  

Optimization of SRAP-PCR System for Valeriana officinalis var. latifolia and Primer Screening

作　　者：梁芳瑜 何茂秋 徐昌艳 杨小生 杨娟[2,3] 罗忠圣 覃容贵 LIANG Fang-yu;HE Mao-qiu;XU Chang-yan;YANG Xiao-sheng;YANG Juan;LUO Zhong-sheng;QIN Rong-gui(School of Pharmaceutical Sciences,Guizhou Medical University,Guiyang 550025,China;The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences,Guiyang 550014,China;State Key Laboratory of Function and Applications of Medicinal Plants,Guiyang 550014,China)

机构地区：[1]贵州医科大学药学院,贵阳550025 [2]贵州省中国科学院天然产物化学重点实验室,贵阳550014 [3]省部共建药用植物功效与利用国家重点实验室,贵阳550014

出　　处：《中国实验方剂学杂志》2021年第23期163-171,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：贵州省教育厅青年科技人才成长项目(黔教科合KY字[2018]183);贵州省教育厅高等学校特色重点实验室平台项目(黔教合KY字[2020]018号);2019年大学生创新创业训练计划项目(20195200144);贵州省发改委工程中心项目(黔财建[2019]303号)。

摘　　要：目的:建立宽叶缬草的相关序列扩增多态性(SRAP)-聚合酶链式反应(PCR)反应体系,为宽叶缬草良种选育提供理论和技术基础。方法:采用单因素试验考察Taq Mix用量,Mg^(2+)浓度,模板DNA浓度,Taq DNA聚合酶浓度对宽叶缬草SRAP-PCR扩增结果的影响,以此为基础进行正交试验,优化宽叶缬草SRAP-PCR反应体系,并在最优反应体系条件下筛选可用于宽叶缬草遗传多样性研究的有效引物。结果:单因素实验结果表明Taq Mix用量为8~11μL时扩增效果较佳;加入低浓度Mg^(2+)可获得较佳扩增效果;中低浓度模板DNA用量可提高扩增效果;加入少量Taq DNA聚合酶可增加扩增结果丰富度。正交实验结果表明各因素对宽叶缬草SRAP-PCR扩增结果的影响程度大小依次为Taq Mix用量>Taq DNA聚合酶加入量>Mg^(2+)加入量>模板DNA用量。适于宽叶缬草的最优反应体系为Taq Mix 11μL,模板DNA 30 ng,Mg^(2+)0.025 mmol·L^(-1),Taq DNA1.5 U,正向引物与反向引物各5μmol·L^(-1),用双蒸水补足体系至20μL。最优退火温度为36.8℃。应用最优体系从88对引物中筛选出17对适用于宽叶缬草SRAP-PCR的条带清晰、多态性较高的有效引物。结论:建立的宽叶缬草SRAP-PCR反应体系稳定性良好,可用于宽叶缬草遗传多样性研究。Objective:To establish the sequence-related amplified polymorphism(SRAP)-polymerase chain reaction(PCR)system for Valeriana officinalis var.latifolia,so as to lay the theoretical and technica foundations for the breeding of V.officinalis var.latifolia.Method:Single factor test was applied to investigate the effects of Taq Mix dose,Mg^(2+)concentration,template DNA concentration,and Taq DNA polymerase conten on SRAP-PCR amplification of V.officinalis var.latifolia,based on which the orthogonal experiments were performed to optimize the SRAP-PCR system for V.officinalis var.latifolia.The effective primers that could be used for genetic diversity studies of V.officinalis var.latifolia were selected under the optimal reaction condition.Result:The results of the single factor test showed that Taq Mix dose within the range of 8-11μL resulted in better amplification.The addition of a low concentration of Mg^(2+),the medium to low concentrations of template DNA,or the low concentration of Taq DNA polymerase enhanced the amplification efficiency or richness.As demonstrated by the orthogonal experiments,the influencing degrees of related factors on SRAP-PCR amplification of V.officinalis var.latifolia were sorted in a descending order as follows:Taq Mix dose>Taq DNA polymerase content>Mg^(2+)concentration>template DNA concentration.The optimal reaction system for V.officinalis var.latifolia was determined to consist of 11μL of Taq Mix,30 ng of template DNA,0.025 mmol·L^(-1)Mg^(2+),1.5 U of Taq DNA polymerase,5μmol·L^(-1)forward primer,and 5μmol·L^(-1)reverse primer,which was supplemented to 20μL with ddH;O.The optimal annealing temperature was 36.8℃.A total of 17pairs of effective primers with high band resolution and polymorphism were selected from 88 primer pairs for SRAP-PCR of V.officinalis var.latifolia.Conclusion:The established SRAP-PCR system for V.officinalis var.latifolia is stable,which can be used for genetic diversity studies of V.officinalis var.latifolia.

关 键 词：宽叶缬草 相关序列扩增多态性(SRAP) 体系优化 引物筛选 正交试验设计 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学]