中国生化药物杂志
中國生化藥物雜誌
중국생화약물잡지
CHINESE JOURNAL OF BIOCHEMICAL PHARMACEUTICS
2015年
7期
17-21
,共5页
张馨元%赵超越%侯和胜%佟少明Δ
張馨元%趙超越%侯和勝%佟少明Δ
장형원%조초월%후화성%동소명Δ
DNA条形码%DNA提取%甘草%黄柏%管花肉苁蓉%肉苁蓉
DNA條形碼%DNA提取%甘草%黃柏%管花肉蓯蓉%肉蓯蓉
DNA조형마%DNA제취%감초%황백%관화육종용%육종용
DNA barcoding%DNA extraction%Glycyrrhiza uralensis Fisch%Phellodendron Chinensis Cortex%Cistanche tubulosa wight%Cistanche deserticola Ma
目的：确定能够满足中药材DNA条形码研究的最适DNA提取方法。方法以中药材甘草、黄柏、管花肉苁蓉和肉苁蓉为实验对象，采用改良高盐低pH法、改良SDS法、CTAB法、PVP法、PlantZol试剂盒及Ezup柱式试剂盒6种DNA提取方法进行DNA提取，采用紫外分光光度法、琼脂糖凝胶电泳和ITS2与psbA－trnH序列的引物PCR扩增对DNA产量及质量进行检测。结果改良高盐低pH法和Ezup柱式试剂盒提取的4种中药材的DNA质量相对较好，其OD260／OD280的值在1．7～1．9之间，PlantZol试剂盒法所提取的DNA得率相对较高，其次为改良高盐低pH法（P＜0．05），但PlantZol试剂盒所提取DNA的纯度较差，DNA电泳检测表明改良高盐低pH法、改良SDS法、CTAB法和PlantZol试剂盒所提取到甘草和管花肉苁蓉的DNA完整性较好，而6种方法所提取的黄柏与肉苁蓉的DNA则均在泳道内呈弥散状态，但只有改良高盐低pH法其ITS2和psbA－trnH序列的引物PCR扩增成功率为100％。结论改良高盐低pH值法提取的基因组DNA可以用来中药DNA条形码的建立，且该方法具有成本低、步骤简单、时间短等优点，是一种高效、快速、经济的中药材基因组DNA提取方法。
目的：確定能夠滿足中藥材DNA條形碼研究的最適DNA提取方法。方法以中藥材甘草、黃柏、管花肉蓯蓉和肉蓯蓉為實驗對象，採用改良高鹽低pH法、改良SDS法、CTAB法、PVP法、PlantZol試劑盒及Ezup柱式試劑盒6種DNA提取方法進行DNA提取，採用紫外分光光度法、瓊脂糖凝膠電泳和ITS2與psbA－trnH序列的引物PCR擴增對DNA產量及質量進行檢測。結果改良高鹽低pH法和Ezup柱式試劑盒提取的4種中藥材的DNA質量相對較好，其OD260／OD280的值在1．7～1．9之間，PlantZol試劑盒法所提取的DNA得率相對較高，其次為改良高鹽低pH法（P＜0．05），但PlantZol試劑盒所提取DNA的純度較差，DNA電泳檢測錶明改良高鹽低pH法、改良SDS法、CTAB法和PlantZol試劑盒所提取到甘草和管花肉蓯蓉的DNA完整性較好，而6種方法所提取的黃柏與肉蓯蓉的DNA則均在泳道內呈瀰散狀態，但隻有改良高鹽低pH法其ITS2和psbA－trnH序列的引物PCR擴增成功率為100％。結論改良高鹽低pH值法提取的基因組DNA可以用來中藥DNA條形碼的建立，且該方法具有成本低、步驟簡單、時間短等優點，是一種高效、快速、經濟的中藥材基因組DNA提取方法。
목적：학정능구만족중약재DNA조형마연구적최괄DNA제취방법。방법이중약재감초、황백、관화육종용화육종용위실험대상，채용개량고염저pH법、개량SDS법、CTAB법、PVP법、PlantZol시제합급Ezup주식시제합6충DNA제취방법진행DNA제취，채용자외분광광도법、경지당응효전영화ITS2여psbA－trnH서렬적인물PCR확증대DNA산량급질량진행검측。결과개량고염저pH법화Ezup주식시제합제취적4충중약재적DNA질량상대교호，기OD260／OD280적치재1．7～1．9지간，PlantZol시제합법소제취적DNA득솔상대교고，기차위개량고염저pH법（P＜0．05），단PlantZol시제합소제취DNA적순도교차，DNA전영검측표명개량고염저pH법、개량SDS법、CTAB법화PlantZol시제합소제취도감초화관화육종용적DNA완정성교호，이6충방법소제취적황백여육종용적DNA칙균재영도내정미산상태，단지유개량고염저pH법기ITS2화psbA－trnH서렬적인물PCR확증성공솔위100％。결론개량고염저pH치법제취적기인조DNA가이용래중약DNA조형마적건립，차해방법구유성본저、보취간단、시간단등우점，시일충고효、쾌속、경제적중약재기인조DNA제취방법。
Objective To establish an optimum DNA extraction method for Chinese traditional medical herbs in order to meet necessary for DNA barcoding research.Methods Four Chinese traditional herbs, Glycyrrhiza uralensis Fisch, Phellodendron Chinensis Cortex, Cistanche tubulosa Wight and Cistanche deserticola Ma were chosen as the experimental materials, the DNA was extracted by 6 different kinds of DNA extraction method, including the improved method of high-salt combined low-pH,the improved method of SDS,CTAB method,PVP method,PlantZol Kit and Ezup Kit, the quality of DNA was investigated by ultraviolet spectrophotometry,agarose gel electrophoresis and PCR amplification by using specific primers of ITS2 and psbA-trnH. ResuIts The quality of the DNA was better than other four kinds of methods by the improved method of high-salt combined low-pH and Ezup Kit, the value of OD260/OD280 was between 1.7 ~1.9,the yield of DNA was the highest by the PlantZol kit , followed by the improved method of high-salt combined low-pH( P <0.05 ) , but the purity of DNA was poor by the PlantZol kit.The DNA electrophoresis tests showed that the DNA integrity of Glycyrrhiza uralensis Fisch and Cistanche tubulosa Wight were better with the improved method of high-salt combined low-pH, the improved SDS method, the CTAB method and the PlantZol kit.The DNA of Phellodendron Chinensis Cortex and Cistanche deserticola Ma were extracted by the six methods appeared diffuse status in the lanes.But only the improved method of high-salt combined low-pH could make the PCR amplification of the success rate 100% by using specific primers of ITS2 and trnH-psbA.ConcIusion The DNA extraction method of high-salt combined low-pH can be used to establish the Chinese DNA barcoding which has the advantages of lower cost, simpler procedure and less time.
