醉马草DNA提取方法的对比与优化  

The Comparison and Optimization of DNA Extraction Methods from Achnatherum inebrians (Drunken horse grass)

作　　者：陈雅琦 苏楷淇 李春杰[1] Chen Yaqi;Su Kaiqi;Li Chunjie(State Key Laboratory of Grassland Agro-ecosystems/Engineering Research Center of Grassland Industry,Ministry of Education/Gansu Tech Innovation Centre of Western China Grassland Industry/Center for Grassland Microbiome/College of Pastoral Agriculture Science and Technology,Lanzhou University,Lanzhou 730000,China)

机构地区：[1]兰州大学草地农业生态系统国家重点实验室/兰州大学草地农业教育部工程研究中心/兰州大学甘肃省西部草业技术创新中心/兰州大学草地微生物研究中心/兰州大学草地农业科技学院,甘肃兰州730000

出　　处：《草学》2020年第5期32-38,共7页Journal of Grassland and Forage Science

基　　金：国家“973”项目课题(2014CB138702);青藏高原第二次科考项目(2019QZKK0302);教育部创新团队发展计划(IRT_17R50);中央高校基本科研业务费项目(LZUJBKY-2020-kb10)资助。

摘　　要：醉马草是一种多年生牧草,为禾本科芨芨草属,常用于机场草坪建植和草原生态恢复,因其重要的经济与生态价值近年来被广泛研究关注。醉马草幼苗含有的酚类、蛋白质等杂质严重影响DNA的提取质量,常规技术不能很好地满足高质量的实验要求。本实验以醉马草幼苗为材料,分别采用SDS、CTAB、高盐法提取醉马草幼苗全基因组DNA。研究发现使用SDS法提取的DNA浓度为176ng·μL^-1,提取率为30%,A值为1.7。使用CTAB法提取的DNA浓度为140ng·μL^-1,提取率为23%,A值为1.68。使用高盐法提取的DNA浓度为37.7ng·μL^-1,提取率为6%,A值为1.1。利用CTAB对提取出的DNA进一步纯化,用琼脂糖凝胶电泳和超微量分光光度计检测DNA的浓度和纯度,通过PCR分析验证优化后CTAB法提取DNA的效果。结果表明各方法纯化后的DNA浓度为:SDS法(223ng·μL^-1)﹥CTAB法(219ng·μL^-1)﹥高盐法(77ng·μL^-1);A值为:SDS法(1.8)>CTAB法(1.77)﹥高盐法(1.4),其中DNA浓度较纯化前分别提高27.1%(SDS法)、57.1%(高盐法)、51.0%(CTAB法),且电泳图片显示DNA条带清晰明亮,无明显拖尾现象。因此,三种方法间使用SDS法提取DNA的效果最佳,且加入纯化步骤可进一步提高DNA提取质量。本研究旨在为醉马草的分子生物学研究提供技术支持与科学依据。Achnatherum inebrians(drunken horse grass)is a kind of perennial grasses,belongs to the subfamily Pooideae,which are commonly utilized as airport lawn and grassland ecological recovery plants.Because of its high value in ecological and economical fields,A.inebrians has been widely attended recently.drunken horse grass seedlings content phenols impurities and protein severely affect the quality of DNA extraction,conventional technology is not able to satisfy the requirement of the high quality on experiment.The experiment applied A.inebrains seedlings as materials,by using SDS,CTAB and high-salt extracted methods respectively to extract drunken horse grass whole DNA.Our study has found that the concentration of DNA was 176 ng·μL^-1,the extracted rate was 30%and A value was 1.7 by using SDS method;the concentration of DNA was 140 ng·μL^-1,the extracted rate was 23%and A value was 1.68 by using CTAB method;and the concentration of DNA was 37.7 ng·μL^-1,the extracted rate was 6%and A value was 1.1 by using high-salt method.What’s more,after purification by CTAB,then tested the quality of DNA by agarose gel electrophoresis and ultramacho spectrophotometer,through PCR analyzed showed that the result has optimized.The concentration of DNA has been SDS(223 ng·μL^-1)﹥CTAB(219 ng·μL^-1)>high-salt(77 ng·μL^-1);A value:SDS(1.8)﹥CTAB(1.77)﹥high-salt(1.4),and concentration had risen 27.1%(SDS)、57.1%(high-salt)、51.0%(CTAB),respectively,and DNA electrophoresis image showed a clear and bright stripe with no obvious trailing phenomenon.Therefore,the SDS method had been the best choose for extracting A.inebrains DNA from the three methods,and further purification by CTAB could increase both quality and quantity.Our study is concentrated on the technique support and theory evidence for the further studies on A.inebrains molecular research.

关 键 词：醉马草 DNA提取质量 SDS法 CTAB法 高盐法 纯化 

分 类 号：S543.03[农业科学—作物学]